Effect of ovarian suppression on glucose metabolism of young lean women with and without ovarian hyperandrogenism

A. Cagnacci1,3, A.M. Paoletti2, S. Arangino1, G.B. Melis2 and A. Volpe1

1 Institute of Obstetrics and Gynaecology of Modena, via del Pozzo 71, 41100 Modena and 2 Institute of Obstetrics and Gynaecology of Cagliari, via Ospedale 46, 09124 Cagliari, Italy


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 References
 
Gonadal steroids are believed to influence glucose metabolism, oestrogens inducing an improvement and androgens or progestins a deterioration. At baseline and after 3 months of ovarian suppression with a gonadotrophin-releasing hormone analogue (GnRHa: goserelin depot 3.75 mg/28 days), glucose metabolism was evaluated in eight lean women affected by ovarian hyperandrogenism (PCOS) and six age–weight-matched non-hyperandrogenic women (controls) by using both an oral glucose tolerance test (75 g; OGTT) and the minimal model method. The latter method allows calculation of peripheral insulin sensitivity (Si) and glucose dependent glucose utilization (Sg). In PCOS, higher fasting concentrations (P < 0.05) of insulin and C-peptide, and lower Sg (P < 0.05) and Si (P < 0.01) were found. GnRHa did not significantly modify glucose metabolism of controls, while in women with PCOS it decreased fasting glucose (P < 0.05) and significantly increased Si (P < 0.03) up to control values. The present data indicate that strong suppression of ovarian activity improves Si in lean women with PCOS, while it is without relevant effects on glucose metabolism of non-hyperandrogenic women.

Key words: C-peptide/GnRHa/insulin/ovary/PCOS


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 References
 
Glucose metabolism and insulin sensitivity are believed to be influenced by gonadal steroids (Cohen and Hickman, 1987Go; Diamond et al., 1989Go; Mortola and Yen, 1990Go; Cagnacci et al., 1992Go, 1998Go; Dunaif et al., 1992Go; Holmang et al., 1992Go; Elkind-Hirsch et al., 1993Go; Godsland et al., 1993Go; Lindheim et al., 1993Go). Several reports have documented positive effects of low doses of oestrogens on glucose metabolism and insulin sensitivity of hypogonadal women, such as postmenopausal women (Cagnacci et al., 1992Go, 1998Go; Elkind-Hirsch et al., 1993Go; Godsland et al., 1993Go; Lindheim et al., 1993Go). On the other hand, androgens are believed to deteriorate glucose metabolism, exogenous administration of androgens being associated with a decrease in peripheral tissue sensitivity to insulin (Cohen and Hickman, 1987Go; Mortola and Yen, 1990Go; Holmang et al., 1992Go; Polderman et al., 1994Go). On these bases, it has been postulated that, in women with ovarian hyperandrogenism, as in those with polycystic ovary syndrome (PCOS), the enhanced levels of androgens may induce insulin resistance and in this way contribute to the perpetuation of the syndrome. However, studies evaluating the role of androgen suppression on glucose metabolism of women with PCOS have obtained conflicting results, and either no effect (Geffner et al., 1986Go; Dunaif et al., 1990Go; Gadir et al., 1990Go; Lanzone et al., 1990Go; Dale et al., 1992) or improvements (Moghetti et al., 1996Go; Dahlgren et al., 1998Go) have been reported. Differences in body mass index, degree of ovarian suppression and experimental settings may be implicated in the contradictory results obtained. Furthermore, the gonadotrophin-releasing hormone analogue (GnRHa)-induced reduction of gonadal oestrogens by eliminating their possible beneficial effects on glucose metabolism may have counterbalanced the eventual advantages deriving from androgen suppression. In the present study, the effect exerted by marked ovarian suppression on glucose metabolism of lean women with PCOS and age- and weight-matched non-hyperandrogenic controls was investigated.

Materials and methods
Each woman gave written informed consent to the study which was previously approved by our local ethical committee. Eight lean [body mass index (BMI) = 22.8 ± 0.25] women, 25.1 ± 1.8 years of age, suffering from PCOS and six age- (24.8 ± 1.7 years of age) and weight- (BMI = 22.5 ± 0.18) matched non-hyperandrogenic women with normal ovarian function and symptomatic uterine leiomyomata or endometriosis were enrolled. PCOS was defined as persistent amenorrhoea or oligomenorrhoea of perimenarchal onset, with three or more of these features: ratio luteinizing hormone (LH)/follicle stimulating hormone (FSH) >1.5, ovarian hyperandrogenism as defined by high levels of total testosterone, free testosterone or androstenedione, Ferriman and Gallwey hirsutism score >10, ultrasound evidence of PCOS (Paoletti et al., 1995Go). Each woman was instructed to consume >200 g/day carbohydrate in the 3 days before testing. Following an overnight fast of 12 h, each woman was admitted to the hospital at 0700 h on 2 consecutive days. Glucose metabolism was investigated by both an oral glucose tolerance test (OGTT) and the minimal model method approach, based on analysis of a frequently sampled i.v. glucose tolerance test (FSIGT) (Welch et al., 1990Go) performed on 2 consecutive days in a randomized order.

For the OGTT, a polyethylene catheter inserted in an antecubital vein was kept patent by a slow infusion of saline solution. A glucose load of 75 g was given orally at 0900 h. Samples of arterialized blood (i.e. venous blood with glucose content similar to that of arterial blood), obtained by forearm warming, were collected at times –30, 0, 15, 30, 60, 90, 120 and 180 min after glucose administration.

For the FSIGT, two polyethylene catheters placed in two antecubital veins were kept patent by a slow infusion of saline solution. One catheter was used for i.v. glucose or insulin administration and the other one for blood collection. At 0900 h, glucose (0.3 g/kg) was injected over 1 min i.v. and was followed 20 min later by an i.v. insulin bolus (Actrapid H-M; Novo Nordisk, SpA, Rome, Italy; 0.03 IU/kg). Samples of arterialized blood were collected at time –15, –10, –5, –1, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 20, 22, 23, 24, 25, 27, 30, 40, 60, 70, 80, 90, 100, 120, 160 and 180 min after glucose load.

Each woman was assigned to receive every 28 days for three cycles a s.c. implant of goserelin depot (3.6 mg Zoladex; Zeneca, Milan, Italy), and at the end of treatment was submitted to the same investigations.

Blood samples, collected on ice into heparinized glass tubes, were immediately centrifuged at 1500 g for 15 min. An aliquot of plasma was immediately tested for glucose levels, while another aliquot was immediately frozen to –25°C until assayed. As in previous studies (Cagnacci et al., 1998Go), blood glucose was assayed by an autoanalyser using the glucose oxidase colorimetric method. Insulin levels were assayed in duplicate in all samples by a radioimmunoassay method using commercial kits (Biodata; Guidonia Montecelio, Roma, Italy), with intra- and inter-assay coefficients of variation (CV) of 6.2 and 7% respectively, and sensitivity of 14.35 pmol/l. C-Peptide levels were analysed in duplicate in all OGTT samples, and in the samples collected in the first 20 min of FSIGT by commercial radioimmunoassay kits (Biodata) with intra- and inter-assay coefficients of variation of 3.2 and 8.5% respectively, and sensitivity of 33.1 pmol/l (Cagnacci et al., 1998Go). Circulating levels of LH, FSH, prolactin, oestradiol, total testosterone, free testosterone, androstenedione and dehydroepiandrosterone sulphate (DHEAS) were also analysed in baseline samples by radioimmunoassay (Paoletti et al., 1995Go).

Responses of glucose, insulin and C-peptide observed during OGTT and in the first 20 min of FSIGT, were reported as absolute values and as area under the curve, calculated by the trapezoid method and expressed in arbitrary units (nmol/l or pmol/lxmin; AUC). In order to have an index of hepatic insulin clearance, the C-peptide/insulin ratio of absolute and integrated values was also calculated (Cagnacci et al., 1992Go, 1998Go). Glucose and insulin values obtained during FSIGT were used to calculate by a computerized algorithm (MINMOD) the sensitivity of glucose elimination to insulin (Si) that is inversely related to insulin resistance. Si is defined as the increase in fractional glucose disappearance due to an increment in plasma insulin, i.e. insulin action (independent of both glucose and insulin levels). Si from minimal model approaches is comparable to values from clamp techniques (Beard et al., 1986Go; Bergman et al., 1987Go). Glucose-dependent glucose elimination (Sg) was defined as the total effect of glucose on fractional glucose disappearance independent of an increase in insulin, but including the contribution of basal insulin (Welch et al., 1990Go). Si was expressed in unitsx10–4/minxµ/ml, and Sg in unitsx10–4/min.

Statistical analysis of the results was performed by the t-test for paired data, or Wilcoxon test, as specified. Two-way analysis of variance (ANOVA) for repeated measures (treatmentxtime, with subjects as replicates) was also used to evaluate differences on glucose or hormone responses to OGTT or first 20 min of FSIGT. All the results are expressed as the mean ± standard error (SE).


    Results
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 Abstract
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 Results
 Discussion
 References
 
Circulating levels and modifications during treatment of LH, FSH, prolactin, oestradiol, total testosterone, free testosterone, androstenedione and DHEAS of controls and women with PCOS are reported on Table IGo. During the administration of goserelin depot, a significant decline in ovarian androgens and oestrogens was observed both in PCOS and control women.


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Table I. Mean ± SE concentrations of LH, FSH, prolactin, oestradiol, total testosterone, free testosterone, androstenedione and dehydroepiandrosterone sulphate (DHEAS) in six non-hyperandrogenic women and eight women with polycystic ovary syndrome (PCOS), before and during the third cycle of gonadotrophin-releasing hormone analogue (GnRHa) administration (goserelin depot 3.6 mg/28 days)
 
In control women, goserelin depot induced no modification in the fasting concentrations of glucose (4.2 ± 0.05 versus 4.5 ± 0.2 nmol/l), insulin (64 ± 12 versus 47 ± 6 nmol/l), C-peptide (275 ± 64 versus 287 ± 56 nmol/l) and the C-peptide/insulin ratio (4.3 ± 0.4 versus 6.8 ± 1.6 nmol/l). Responses to OGTT were not significantly different (Figure 1Go). In addition, integrated responses to OGTT of glucose (1020 ± 52 and 1036 ± 128 AUC), insulin (85 359 ± 24 228 versus 51 836 ± 5584), C-peptide (18 648 ± 62 585 versus 192 346 ± 53 691 AUC), and C-peptide/insulin (2.2 ± 0.5 and 4.0 ± 1.2 AUC) were not significantly modified. Similar results were obtained by evaluating the responses to the i.v. glucose administration in the first 20 min following the i.v. bolus (Figure 2Go). No significant variation was observed in the integrated response of glucose (203 ± 13 versus 189 ± 15 AUC), insulin (6371 ± 1172 versus 5378 ± 811 AUC), C-peptide (10 453 ± 2956 versus 10 619 ± 2174 AUC), and C-peptide/insulin ratio (1.8 ± 0.5 versus 2.2 ± 0.5 ). Sg was not significantly modified (0.04 ± 0.007 versus 0.04 ± 0.006) as well as Si (5.97 ± 0.93 versus 4.62 ± 1.53) (Figure 3Go).



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Figure 1. Mean (± SE) glucose, insulin, C-peptide, C-peptide/insulin responses to an oral glucose tolerance test observed in six non-hyperandrogenic controls (left) and in eight hyperandrogenic women with polycystic ovary syndrome (PCOS) (right), prior to (open circles) and after three cycles (closed circles) of administration of the gonadotrophin-releasing hormone analogue (GnRHa) goserelin depot (3.6 mg/28 days). Arrows indicate time of glucose administration.

 


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Figure 2. Mean (± SE) glucose, insulin, C-peptide, C-peptide/insulin responses in the first 20 min following i.v. administration of glucose (30 mg/kg), observed in six non-hyperandrogenic controls (left) and in eight hyperandrogenic women with polycystic ovary syndrome (PCOS) (right) prior to (open circles) and after three cycles (closed circles) of administration of the gonadotrophin-releasing hormone analogue (GnRHa) goserelin depot (3.6 mg/28 days). Arrows indicate time of glucose administration.

 


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Figure 3. Mean (± SE) glucose-dependent glucose elimination (Sg; unitsx10–4/min) (left) and peripheral insulin sensitivity of glucose elimination to insulin (Si; unitsx10–4/minxµ/ml) (right) observed in six non-hyperandrogenic controls and in eight hyperandrogenic women with polycystic ovary syndrome (PCOS) prior to (open bars) and after three cycles (hatched bars) of administration of the gonadotrophin-releasing hormone analogue (GnRHa) goserelin depot (3.6 mg/28 days). *P < 0.05, ***P < 0.01 versus controls; §P < 0.05 versus before.

 
In women with PCOS, goserelin depot induced a decline in fasting levels of glucose (4.3 ± 0.3 versus 3.8 ± 0.2 nmol/l; P < 0.05) but not of insulin (97 ± 17 versus 88 ± 24 nmol/l), C-peptide (573 ± 108 versus 552 ± 146 nmol/l) and C-peptide/insulin ratio (7.8 ± 2.2 versus 8.5 ± 2.9 nmol/l). Responses to OGTT were not significantly different, although they tended to be lower after treatment (Figure 1Go). Integrated responses to OGTT of glucose (1189 ± 335 versus 913 ± 59 AUC), insulin (90 652 ± 34 751 versus 74 913 ± 19 254 AUC), C-peptide (423 006 ± 131 336 versus 294 219 ± 26 294 AUC), and C-peptide/insulin (6.4 ± 1.5 versus 5.8 ± 1.4 AUC) were not significantly modified. Similar results were obtained by evaluating responses to i.v. glucose administration in the first 20 min following the i.v. glucose bolus (Figure 2Go). No difference was observed in the integrated response of glucose (194 ± 5 versus 178 ± 14 AUC), insulin (13 062 ± 2660 versus 11 280 ± 3248 AUC), C-peptide (4903 ± 1451 versus 3771 ± 963 AUC) and C-peptide/insulin ratio (0.49 ± 0.15 versus 0.52 ± 0.13). Sg was not significantly modified (0.023 ± 0.002 versus 0.028 ± 0.004), while Si was significantly increased (3.16 ± 0.46 versus 5.23 ± 1.62; P < 0.03) (Figure 3Go).

In comparison to the control group, women with PCOS showed higher levels (P < 0.05) of insulin and C-peptide in fasting conditions, a higher C-peptide/insulin ratio at the OGTT (P < 0.05), and a lower C-peptide/insulin ratio at the FSIGT (P < 0.02). Sg was lower in women with PCOS (P < 0.05). Also, Si was lower in women with PCOS prior to treatment (P < 0.01), but not during treatment.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 References
 
As previously reported, administration of a GnRHa for 3–6 months (Buyalos et al., 1997Go; Dahlgren et al., 1998Go), but not for 14 days (Vrtacnik-Bokal and Meden-Vrtovec, 1998Go) was capable of bringing circulating concentrations of ovarian androgens within the range of non-hyperandrogenic women. Sex-hormone-binding-globulins were not analysed, but in a previous study on PCOS their concentrations were not modified by the administration of the same GnRHa, goserelin depot, for 6 months (Dahlgren et al., 1998Go).

In women with PCOS, a simple evaluation of glucose–insulin metabolism via an OGTT failed to show both herein and previously (Geffner et al., 1986Go; Gardir et al., 1990; Lanzone et al., 1990Go; Dale et al., 1992), a relevant modification of glucose metabolism during the suppression of ovarian activity. However, by using the minimal model method, it was observed that the reduced Si of lean PCOS was almost completely abolished by suppression of ovarian function via the prolonged administration of a GnRHa. These results are in accordance with recent data (Moghetti et al., 1996Go; Dahlgren et al., 1998Go) in which insulin sensitivity was evaluated by the hyperinsulinaemic glucose clamp technique, in a population of women with normal to moderately increased BMI, and are at variance with those of Dunaif et al. (1990) obtained with an incomplete pituitary desensitization in overweight to frankly obese PCOS.

A reduction of Si does not seem to be the only abnormality of glucose metabolism present in the lean women with PCOS in this study, and, in accordance with what has previously been found in overweight women with PCOS, a reduction in Sg was also present (Falcone et al., 1992Go). In support of its different and independent regulation (Falcone et al., 1992Go) Sg, in contrast to Si, was not modified by ovarian suppression. Because alterations of glucose dependent glucose transport have not been observed in in-vitro cells of women with PCOS (Ciaraldi et al., 1992), the mechanisms implicated in Sg alteration are unclear. However, a decrease in Sg may favour enhanced insulin secretion from pancreatic ß-cells (Holte et al., 1994Go; Ciampelli et al., 1998Go) and contribute to generate or perpetuate hyperinsulinaemia and peripheral insulin resistance of PCOS (Dunaif, 1997Go; Nestler and Jakubowicz, 1997Go; Sattar et al., 1998Go).

Hepatic insulin clearance, roughly evaluated by the integrated C-peptide/insulin ratio during i.v. glucose administration, was lower in PCOS than in controls. This alteration was not likely to be dependent on hyperandrogenism, which does not seem to influence hepatic insulin sensitivity (Peiris et al., 1989Go; Dunaif et al., 1990Go; Moghetti et al., 1996Go), and indeed hepatic insulin clearance was not modified by ovarian suppression. On the other hand, when the C-peptide/insulin ratio was evaluated during OGTT, it was not reduced and indeed was enhanced in women with PCOS. Because oral glucose administration stimulates gastrointestinal factors, such as incretins (Shapiro et al., 1987Go), capable of influencing hepatic insulin clearance (Shuster et al., 1988Go), it is likely that this stimulus is modified in women with PCOS. Indeed, some gastrointestinal mechanisms involved in glucose metabolism are sensitive to sex steroids (Cagnacci et al., 1998Go). Additionally, in PCOS fasting beta-endorphin levels are elevated (Givens et al., 1987Go) and are further stimulated by oral glucose administration (Laaitikainen et al., 1989; Carmina et al., 1992Go). Because beta-endorphin is believed to influence glucose–insulin metabolism (Givens et al., 1987Go; Fulghesu et al., 1995Go), its modifications may also play a role in the complex modulation of hepatic insulin clearance of PCOS.

A surprising finding of the present study was the failure of the marked ovarian suppression induced by the GnRHa to modify glucose metabolism of non-hyperandrogenic women. This seems at variance with data suggesting that hypo-oestrogenism may impair glucose metabolism in postmenopausal women (Cagnacci et al., 1992Go, 1998Go; Elkind-Hirsch et al., 1993Go; Godsland et al., 1993Go; Lindheim et al., 1993Go). However, in postmenopausal women, as the consequence of ageing (Gumbiner et al., 1989Go; Shimokata et al., 1991Go), glucose metabolism may be more critically regulated, and modifications of gonadal steroids may have a more dramatic impact than in young lean women. Furthermore, hypo-oestrogenism induced by the GnRHa administration is different from that of the menopause. GnRHa may exert direct effects on insulin clearance, which can mask eventual negative effects of hypo-oestrogenism (Dunaif et al., 1990Go). Furthermore, the administration of GnRHa is associated with a marked decline in ovarian androgens, as well as with a reduction in growth hormone levels (Kaltsas et al., 1998Go) which may counterbalance the negative effect of hypo-oestrogenism on glucose metabolism. Whatever the mechanism is, the present data indicate that at least in young lean women, the administration of GnRHa has no negative effect on glucose metabolism, and this is reassuring for the prolonged administration of these compounds.


    Notes
 
3 To whom correspondence should be addressed Back


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Submitted on May 22, 1998; accepted on December 16, 1998.