1 Division of Endocrinology and Human Reproduction, Second Department of Obstetrics and Gynecology, and 2 Department of Biochemistry, Medical School, Aristotle University of Thessaloniki, Greece
3 To whom correspondence should be addressed at: Division of Endocrinology and Human Reproduction, Second Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, 119, Mitropoleos Str., 54622, Thessaloniki, Greece. e-mail: argic{at}med.auth.gr
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Abstract |
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Key words: adiponectin/adiposity/body mass index/insulin resistance/PCOS
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Introduction |
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In humans, adiponectin levels were found paradoxically to be decreased in obese, compared with normal individuals (Arita et al., 1999), making it the only known adipocyte-specific hormone that is down-regulated in obesity. Moreover, decreased adiponectin levels are associated with coronary artery disease and increase significantly after weight reduction (Hotta et al., 2000
), whereas high adiponectin levels are independently associated with increased insulin sensitivity (Weyer et al., 2001
; Philips et al., 2003
) and reduced risk of type 2 diabetes in Pima Indians (Lindsay et al., 2002
). Recently, beneficial effects of adiponectin on both glucose and lipid metabolism in white, non-diabetic individuals have been reported. Specifically, plasma adiponectin levels were negatively correlated with 2-h triglycerides and the free fatty acid (FFA) concentrations during the oral glucose tolerance test (OGTT), and correlated positively with high-density lipoprotein (HDL) levels and insulin sensitivity, measured using an euglycaemic clamp and estimated from the OGTT. Those relationships remained significant after adjustments for sex and percentage of body fat (Tschritter et al., 2003
). Furthermore, in another recently published study, adiponectin concentrations in plasma were found to be independently associated with a reduced risk of type 2 diabetes in apparently healthy individuals (Spranger et al., 2003
).
With regard to human reproduction, women were found to have significantly higher adiponectin levels than men (Arita et al., 1999). A similar sexual dimorphism was also demonstrated in mice (Combs et al., 2003
), wherein tissue adiponectin levels were found to rise early in puberty, and to increase further after neonatal castration in males or adult ovariectomy in females. Furthermore, adiponectin expression was found to be suppressed by estrogens in vitro, and stimulated by bromocryptine while being suppressed by prolactin.
All of the above findings favour a pleiotropic insulin-sensitizing action of adiponectin, which seems to antagonize the inflammatory and other humoral factors that promote insulin resistance, and is subject to complex hormonal control. The adiponectin receptor and downstream signalling pathway have been identified (Steppan and Lazar, 2002).
Polycystic ovary syndrome (PCOS) is characterized by chronic anovulation and hyperandrogenism (Zawadski and Dunaif, 1992; Panidis et al., 2000; Costello and Eden, 2003
). PCOS affects 510% of women of reproductive age (Franks, 1995
), and is responsible for 5070% of cases with anovulatory infertility (Adams et al., 1986
; Hull, 1987
). It is, therefore, the most frequent cause of anovulation and probably the most common endocrine disorder among women (Homburg, 1996
).
Insulin resistance with compensatory hyperinsulinaemia are prominent features of PCOS. Both lean and obese women with PCOS show reduced insulin sensitivity and hyperinsulinaemia to some extent (Dunaif et al., 1989), but insulin resistance is exacerbated by the interaction between obesity and the syndrome (Dunaif, 1994
; 1997). Hyperinsulinaemia is thought to result in increased androgen biosynthesis (Adashi et al., 1985
) and decreased levels of sex hormone-binding globulin (SHBG) (Nestler et al., 1991
), playing a major role in the pathogenesis of hyperandrogenism. In addition to reproductive morbidity, insulin resistance and the resultant hyperinsulinism place patients at risk of long-term metabolic disorders, such as impaired glucose tolerance (up to 35%) and type 2 diabetes (up to 10%) (Ehrmann et al., 1999
), as well as cardiovascular disease (Futterweit, 1999
).
The aim of the present study was to: (i) measure serum adiponectin levels in women with PCOS and with a body mass index (BMI) either less than or more than 25 kg/m2; and (ii) assess possible correlations between adiponectin and the hormonal or metabolic parameters of the syndrome. The study was conducted in order to investigate the possible involvement of adiponectin in the pathogenesis of PCOS, since it has been stated that adiponectin might be a possible link between obesity and insulin resistance; moreover, adiponectin seems to interact with the gonadal axis. Finally, PCOS is associated with impaired glucose tolerance, diabetes mellitus and obesity. It should be noted that no previously published studies on circulating adiponectin levels in women with PCOS were identified.
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Materials and methods |
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The women were allocated to three groups on the basis of BMI value and a diagnosis of PCOS. Hence, group I (n = 35) women had PCOS + BMI >25 kg/m2; group II (n = 35) had PCOS + BMI <25 kg/m2; and group III (controls; n = 15) were ovulating without hyperandrogenaemia + BMI <25 kg/m2. A diagnosis of PCOS in groups I and II was based on the presence of chronic anovulation (fewer than six cycles in 12 months) and hyperandrogenaemia. Furthermore, other common causes of hyperandrogenism (prolactinoma, congenital adrenal hyperplasia, Cushing syndrome and virilizing ovarian or adrenal tumours) were excluded, in accordance with National Institutes of Health criteria (Zawadski and Dunaif, 1992). All women in group III had normal ovulating cycles (blood progesterone levels >10 ng/ml in two consecutive cycles), and no signs of hyperandrogenism. In all women, the basal serum levels of FSH, LH, testosterone,
4-androstenedione and dehydroepiandrosterone sulphate (DHEA-S) were measured. Serum levels of prolactin, 17
-OH-progesterone, SHBG, glucose, insulin and adiponectin were also measured. The free androgen index (FAI) was calculated according to the equation: testosterone (nmol/l) x 100/SHBG (nmol/l). The glucose:insulin ratio was also calculated.
Blood samples were collected between days 3 and 6 of a spontaneous menstrual cycle, at 09:00, after an overnight fast. Informed consent was obtained from all 85 women, and the study was approved by the Institutional Review Board.
Assay methods
All assays of hormonal levels and plasma glucose were carried out at the Department of Biochemistry of the Aristotle University of Thessaloniki School of Medicine.
Plasma glucose concentrations were measured using a glucose oxidase technique with an auto analyser (Roche/Hitachi 902; Roche Diagnostics GmbH, Manheim, Germany). LH, FSH and prolactin levels were measured with an enzyme-linked immunoassay (EIA), using commercial kits (Nichols Institute Diagnostics, CA, USA). Testosterone was measured with a Direct RIA kit (Sorin, Biomedica); 4-androstenedione with a Gamma Coat [125I] RIA kit (Incstar Corp.); DHEA-S with direct RIA solid-phase coated tubes (Zer Science Based Industries Ltd); 17
-OH-progesterone with an ImmuChem Double Antibody [125I] RIA kit (ICN Pharmaceuticals, Inc.); insulin with a Coat-A-count Insulin kit (Diagnostic Products Corp.); and SHBG with an immunoradiometric assay (IRMA) kit (SHBG: [125I] IRMA Kit, Orion Diagnostica). Adiponectin levels were measured with a commercial radiometric immunoassay kit (Linco Research Inc., St Charles, MO, USA).
The intra-assay coefficients of variation (CV) were 1.5% for FSH, 0.7% for LH, 2.7% for prolactin, 3.8% for insulin, 4.1% for 17-OH-progesterone, 1.3% for testosterone, 5.9% for androstenedione, 9.4% for DHEA-S, 5.8% for SHBG and 3.6% for adiponectin. The average inter-assay CV were 3.2% for FSH, 1.7% for LH, 3.4% for prolactin, 4.4% for insulin, 6.3% for 17
-OH-progesterone, 2.2% for testosterone, 9.2% for androstenedione, 12.1% for DHEA-S, 7.8% for SHBG and 5.2% for adiponectin.
Statistical analysis
Statistical analyses were performed using SPSS software (v. 11.5 SPSS, Inc., Chicago, IL, USA). All values were log-transformed to achieve a more normal distribution. Means were compared with the unpaired t-test. Bivariate correlation analysis (calculation of the Pearson coefficient) was used to assess the correlation of serum adiponectin levels to each parameter. Independent relationships between serum adiponectin levels and those parameters to which they were found to correlate significantly were assessed using multiple regression and partial correlation analysis (controlling for those parameters that where found to be independent determinants of serum adiponectin levels in multiple regression analysis). A P-value < 0.05 was considered statistically significant.
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Results |
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Serum adiponectin levels in group I were significantly lower than those of groups III and II (P < 0.05), whereas no significant difference existed between women with PCOS and normal BMI and women without PCOS. Serum concentrations of insulin and adiponectin, as well as blood glucose levels and the value of the glucose:insulin ratio and FAI in all three groups are summarized in Figure 1C.
Calculation of the Pearson coefficient showed that adiponectin levels were positively correlated with SHBG levels (r = +0.224, P < 0.05). Serum adiponectin levels were found to be negatively correlated with BMI (r = 0.322, P < 0.005) (Figure 2), and with 4-androstenedione levels (r = 0.327, P < 0.005) (Figure 3), FAI (r = 0.23, P < 0.05) and plasma glucose level (r = 0.242, P < 0.05). In women with PCOS as a group, serum adiponectin levels were also negatively correlated with BMI (r = 0.316, P < 0.01),
4-androstenedione levels (r = 0.335, P < 0.01) and plasma glucose levels (r = 0.277, P < 0.05), whereas in controls adiponectin was not correlated with any parameter. No significant correlation was observed between serum adiponectin levels and the concentration of FSH, LH, testosterone, DHEA-S, 17
-OH-progesterone, insulin or the glucose:insulin ratio. In multiple regression analysis (n = 85), the BMI and
4-androstenedione level were found to be significant independent determinants of adiponectin levels (r2 = +0.1, P < 0.005 and r2 = +0.11, P < 0.005 respectively) (Figures 2 and 3). Partial correlation analysis showed that SHBG levels and FAI did not correlate with circulating adiponectin independently of either BMI or
4-androstenedione level. However, the correlation of serum adiponectin levels with glucose concentration remained significant (P < 0.05) after adjustment for
4-androstenedione levels, but not for BMI.
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Discussion |
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The present study is the first in which serum adiponectin levels in women with PCOS were measured. It is difficult to draw any firm conclusions from these data; nevertheless, adiponectin levels did not differ significantly between women in groups II and III, though women with PCOS and normal BMI had significantly higher levels of insulin, and a smaller glucose:insulin ratioin other words, they were more resistant to insulin. This finding might lead to the conclusion that, in PCOS, adiponectin is not associated with insulin resistance that is not induced by obesity.
Nonetheless, insulin resistance of PCOS and its compensatory hyperinsulinaemia are most marked when there is interaction between the syndrome and obesity (Dunaif, 1994; 1997). Adiponectin levels were significantly lower in women with a BMI >25 kg/m2 than in women with a normal BMI, along with higher insulin levels. The glucose:insulin ratio was significantly smaller in women with a BMI >25 kg/m2, compared with the ratio in women with a normal BMI. Bivariate correlation analysis, however, showed that adiponectin levels did not correlate with either insulin levels or the glucose:insulin ratio, which is an index of insulin resistance (Legro et al., 1998
); rather, they were negatively correlated with BMI, glucose levels and FAI, and positively correlated with SHBG levels. The latter is reduced in PCOS (Figure 1C), and the reduction is further induced by obesity (Nestler et al., 1991
). This relationship also explains the higher levels of circulating androgens (FAI) in women with PCOS and BMI >25 kg/m2, compared with women with PCOS and a BMI <25 kg/m2.
In other words, adiponectin levels were found to correlate significantly with certain obesity-associated parameters. This finding might indicate the involvement of adiponectin in the dysregulated metabolic state of PCOS. Therefore, a stepwise multiple regression analysis and subsequent partial correlation adjusting for independent determinants was performed, in order to assess independent relationships. Notably, adiponectin levels were found not to correlate with any of the above parameters, independently of BMI. These findings do not appear to favour a direct link between adiponectin and PCOS-associated insulin resistance, independently of increased adiposity.
The present results seem to be inconsistent with substantial evidence suggesting that adiponectin plays an important, beneficial role in insulin sensitivity in rodents (Berg et al., 2001; Yamamuchi et al., 2001
), non-human primates (Hotta et al., 2001
) and humans (Hotta et al., 2000
; Weyer et al., 2001
; Philips et al., 2003
; Spranger et al., 2003
; Tschritter et al., 2003
). Based on the present findings alone, it cannot be excluded with certainty that adiponectin is involved in PCOS-associated resistance to insulin. The negative results obtained might be due to the small number of women studied, in addition to the fact that insulin resistance was estimated using the glucose:insulin ratio. Although this ratio has been suggested as a simple and valuable measure of insulin sensitivity in women with PCOS (Legro et al., 1998
), more sensitive (Radziuck, 2000
) and dynamic methods (namely the hyperinsulinaemic isoglycaemic glucose clamp and OGTT) have been implemented in most studies demonstrating the insulin-sensitizing effects of adiponectin in vivo. Notably, however, none of these reports included women with PCOS.
Existent, yet paradoxical, interactions between adiponectin and gonadal function and development in mice have been reported recently (Combs et al., 2003). In the present study, no correlation between serum adiponectin levels and testosteronethe hormone mainly responsible for PCOS-associated clinical hyperandrogenismwas found, and there was no BMI-independent correlation with FAI. However, a significant and independent correlation with
4-androstenedione levels was observed (Figure 3), and this was suggestive of an interaction between adiponectin and steroid synthesis or action. Intriguingly, a strong negative correlation between another hormone (ghrelin) and
4-androstenedionebut not with testosterone and other androgenshas been recently reported (Pagotto et al., 2002
). The correlation with glucose, though BMI-dependent, remained significant after adjustment for
4-androstenedione levelsunlike SHBG levels and FAI, which were not correlated with circulating adiponectin independently of either BMI or
4-androstenedione levels.
In conclusion, the negative correlation between serum adiponectin and BMI may be confirmed for the first time in women with PCOS. It is also evident that women with PCOS and a normal BMI do not have significantly higher serum adiponectin levels than ovulating women with normal BMI and without hyperandrogenism, and that circulating adiponectin levels are independently correlated with 4-androstenedione levels. No correlation between adiponectin levels and the glucose:insulin ratio, insulin, gonadotrophin, testosterone or 17
-OH-progesterone levels was found. Finally, the observed correlation between adiponectin levels and glucose, SHBG and FAI was seen to be BMI-dependent. The above findings suggest that adiponectin is most likely not actively involved in the pathogenesis of PCOS, though it might play a role in the complicated metabolic abnormalities of the syndrome.
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Submitted on March 11, 2003; accepted on May 7, 2003.