1 Division of Endocrinology and Human Reproduction, Second Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, Thessaloniki, 2 Division of Endocrinology, Department of Medicine and 3 Genetics Unit, Department of Obstetrics and Gynecology, University of Ioannina, Ioannina, Greece
4 To whom correspondence should be addressed at: 119 Mitropoleos Street, 54622, Thessaloniki, Greece. Email: argic{at}med.auth.gr
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Abstract |
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Key words: adiponectin/polycystic ovary syndrome/gene polymorphism
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Introduction |
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The infamous metabolic syndrome or X-syndrome, i.e. the combination of insulin resistance with hypertension, dyslipidemia and central obesity, is nowadays regarded as a major risk factor for the development of DM2 and cardiovascular disease (Reusch, 2002). Furthermore, reproductive disorders are often observed in pre-menopausal women with metabolic syndrome. Therefore, the combined metabolic and reproductive morbidity of PCOS has recently led to the introduction of the term XX-syndrome (Sam and Dunaif, 2003
).
Once regarded as an inert depot of triglycerides, adipose tissue has recently emerged as an active endocrine organ that regulates energy metabolism by secreting a number of substances, so-called adipo(cyto)kines (Ahima and Flier, 2000). Apparently, these peptides influence peripheral insulin sensitivity, playing an important role in the pathogenesis of metabolic syndrome (Mantzuzawa et al., 1999
).
One relatively well-studied molecule of this kind is adiponectin, identified in 1995 by Scherer et al. (1995). Adiponectin is the only known adipocytokine, the levels of which are reduced in obese, compared with normal, individuals (Arita et al., 1999
). Adiponectin has been shown to have anti-atherogenic effects (Ouchi et al., 1999
; 2001
; Okamoto et al., 2000
), and a number of studies also suggest a potent insulin-sensitizing action of this hormone (Lindsay et al., 2002
; Daimon et al., 2003
; Spranger et al., 2003
; Tschritter et al., 2003
).
The adiponectin gene consists of three exons and two introns spanning a 17-kb region, and has been located in chromosome 3q27 (Scherer et al., 1995; Hu et al., 1996
). One common and two rare genetic polymorphisms in the adiponectin gene have been identified in non-diabetic populations (Schaffler et al., 2000
; Takahashi et al., 2000
). The silent T/G polymorphism in exon 2 of the human adiponectin gene (T45G
Gly15Gly) could somehow affect plasma adiponectin levels (Takahashi et al., 2000
; Yang et al., 2003a
; b
). This highly prevalent polymorphism has recently been reported to be associated with the risk of obesity, insulin resistance, DM2 and high levels of low-density lipoprotein cholesterol (Hara et al., 2002
; Menzaghi et al., 2002
; Stumvoll et al., 2002
; Ukkola et al., 2003
; Yang et al., 2003a
; b
). In contrast, others have found no association of this particular locus with obesity or DM2 (Schaffler et al., 2000
; Takahashi et al., 2000
; Zietz et al., 2001
; Filippi et al., 2004
).
Despite the numerous reports studying the association between adiponectin gene polymorphisms and insulin resistance or obesity, until now, no studies have examined adiponectin gene polymorphisms in women with PCOS. In this study, we investigated the possible association of the T45G adiponectin gene polymorphism with PCOS.
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Subjects and methods |
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All 232 women were divided into two groups, based on the diagnosis of PCOS: group I, 132 women with PCOS [62 with body mass index (BMI) >25 kg/m2 and 70 with BMI <25 kg/m2]; and group II, 100 ovulating women (19 with BMI >25 kg/m2 and 81 with BMI <25 kg/m2) without hyperandrogenemia (controls). Diagnosis of PCOS in women from group I was based on the presence of chronic anovulation (less than six cycles in 12 months) and hyperandrogenemia. Furthermore, other common causes of hyperandrogenism (prolactinoma, congenital adrenal hyperplasia, Cushing syndrome and virilizing ovarian or adrenal tumours) were excluded, in accordance with the criteria proposed in 1990 by the NIH and revised in 2003 (Zawadski and Dunaif, 1992; The Rotterdam ESHRE/ASRM Sponsored PCOS Consensus Workshop Group, 2004
). All women in group II had normal ovulating cycles (28±2 days, mean±SD) with 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 (
4A) and dehydroepiandrosterone sulfate (DHEA-S) were measured. Serum levels of prolactin (PRL), 17
-OH-progesterone, sex-hormone-binding globulin (SHBG), glucose and insulin were also measured. In 61 women with PCOS and 25 controls, serum adiponectin levels were also available. All measurements were performed between the third and sixth day of the menstrual cycle. Free androgen index (FAI) was calculated according to the equation: testosterone (nmol/l)x100/SHBG (nmol/l). Glucose-to-insulin ratio was also calculated.
Blood samples were collected between the third and sixth day of a spontaneous menstrual cycle, at 09:00, after an overnight fast. Informed consent was obtained from all 232 women and the study was approved by the Institutional Review Board. All women with PCOS were outpatients attending the Endocrine Unit of the 2nd Department of Obstetrics and Gynecology of the Aristotle University of Thessaloniki at the Hippocration Hospital of Thessaloniki. Control women were volunteers. Genotype analysis was performed at the Genetics Unit of the Department of Obstetrics and Gynecology at the University of Ioannina.
Assay methods
Hormonal measurements. The basal serum levels of FSH, LH, testosterone, 4A, DHEA-S, PRL, 17
-OH-progesterone, SHBG, adiponectin, insulin and glucose were measured, as described previously (Panidis et al., 2003
).
Genotype analysis. Genomic DNA was isolated from peripheral blood leukocytes of women with PCOS and the controls. The adiponectin T45G polymorphism, located in exon 2, was genotyped by amplification of genomic DNA using the following primers: F5'-GAATGAGACTCTGCTGAGATGG and R5'- TATCATGTGAGGAGTGCTTGGATG. PCR products were obtained using 25 µl reactions [0.3 µg genomic DNA, 2 pmol/µl primers, 2 mmol/l each of deoxy-ATP, TGP, CTP and TTP, 0.5 U TaqDNA polymerase (Invitrogen, Life Technologies, USA), 1 mmol/l MgCl2] in a thermal cycler (PTC-100; MJ Research, Inc., USA). The amplification conditions were as follows: 94°C for 5 min, followed by 30 cycles of 30 s at 94°C, 30 s at 60°C and 90 s at 72°C, and ending with a single 10 min extension step at 72°C. The resulting fragment was 372 bp in length. The polymorphism was typed with enzyme Smal (BioLabs Inc., New England). Digestion of the G allele produced two fragments with lengths 216 and 156 bp. The digestion products were resolved after electrophoresis in 2% agarose gel (Figure 1).
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Results |
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Discussion |
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The purpose of our study was to examine for the first time: (i) the frequency of the common T45G polymorphism in exon 2 of the adiponectin gene in women with PCOS, compared with normal controls; and (ii) the effect of this polymorphism on anthropometric, hormonal and metabolic parameters of the syndrome. In all groups, no association of T45G polymorphism with BMI and glucose-to-insulin ratio was observed.
This finding is in concordance with recent studies, where no association was found between obesity or insulin resistance and the T45G polymorphism (Schaffler et al., 2000; Takahashi et al., 2000
; Zietz et al., 2001
; Filippi et al., 2004
). Nevertheless, significant associations between this highly prevalent polymorphism and obesity or insulin resistance have also been reported (Hara et al., 2002
; Menzaghi et al., 2002
; Stumvoll et al., 2002
; Ukkola et al., 2003
; Yang et al., 2003a
; b
). Our findings support the hypothesis that the T45G polymorphism in exon 2 of the adiponectin gene is not directly linked to obesity and the metabolic disturbances of the X-syndrome, as demonstrated in PCOS, at least in the population studied.
It is interesting, though, that different frequencies of the TG+GG genotypes were found in women with PCOS compared with controls. Furthermore, the frequency of GG+TG genotypes in a subgroup of PCOS women with relatively higher 4A levels was significantly higher compared with the control group, although no difference was noticed between the subgroup of PCOS women with relatively lower
4A levels and the control group. Testosterone levels and FAI were not correlated with genotype frequencies in a similar way. In a recently published study (Panidis et al., 2003
) it was demonstrated that
4A, but not testosterone, levels were negatively correlated with serum adiponectin levels. Moreover, this correlation was BMI-independent.
Subjects with PCOS carrying the G allele had lower adiponectin levels, but without statistical significance (Figure 2). This finding is in agreement with previous data (Takahashi et al., 2000). Furthermore, a significant correlation of another polymorphism (G276T) with lower circulating adiponectin has been observed (Hara et al., 2002
). The two polymorphisms might be in linkage disequilibrium, and the G/G haplotype has been strongly associated with the metabolic disturbances of PCOS, as well as lower plasma adiponectin concentration (Menzaghi et al., 2002
).
Therefore, although the number of women homozygous for the G allele in our study was relatively small to draw firm conclusions, we postulate that there is a complex relationship, possibly a negative feedback loop, between adiponectin and the hypothalamicpituitarygonadal axis, specifically steroid synthesis or action. Indeed, in vitro studies have shown that both glucocorticoids and androgens (Nishizawa et al., 2002) down-regulate the expression of adiponectin, and there is substantial evidence suggestive of a complex interaction between this hormone and gonadal function (Combs et al., 2003
).
However, in a previous study (Panidis et al., 2003), no difference was found in plasma adiponectin levels between women with PCOS and BMI-matched controls, which is in agreement with another recently published report (Orio et al., 2003
). It was concluded that circulating adiponectin is not actively involved in the metabolic aspects of the syndrome. Nevertheless, it is possible that adipokines also act in a paracrine manner (Panidis et al., 2004a
). For instance, a very recent study suggests that resistin could be involved in the pathogenesis of the syndrome as a local factor (Seow et al., 2004
), although no difference in plasma levels was observed, in concordance with our own report on resistin levels in PCOS (Panidis et al., 2004b
).
In conclusion, our findings suggest an interaction between adiponectin and steroid synthesis or action in PCOS, although the physiological significance of such a relationship remains obscure at present. No statistically significant link between the T45G adiponectin gene polymorphism and circulating adiponectin, obesity or the metabolic aspects of PCOS was observed, although women with the syndrome carrying the G allele had a tendency for lower serum adiponectin levels. Since the T45G polymorphism is a synonymous mutation, the exact molecular mechanisms responsible for the biological effects of this variation are not known at present. It is plausible that this polymorphism is in linkage disequilibrium with some other functional genetic alterations. More data are needed to specify the systemic and local function of the newly identified adipocytokines in the pathophysiology of PCOS.
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References |
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---|
Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K, Shimomura I, Nakamura T, Miyaoka K et al. (1999) Paradoxical decrease of an adipose specific protein, adiponectin in obesity. Biochem Biophys Res Commun 257, 7983.[CrossRef][Medline]
Chandran M, Philips SA, Ciaraldi T and Henry RR (2003) Adiponectin: more than just another fat cell hormone? Diabetes Care 26, 24422450.
Combs TP, Berg AH, Rajala MW, Klebanov S, Iyengar P, Jimenez-Chillaron JC, Patti ME, Klein SL, Weinstein RS and Scherer PE (2003) Sexual differentiation, pregnancy, calorie restriction and aging affect the adipocyte-specific protein adiponectin. Diabetes 52, 268276.
Daimon M, Oizumi T, Saitoh T, Kameda W, Hirata A, Yamaguchi H, Ohnuma H, Igarashi M, Tominaga M and Kato T (2003) Decreased serum levels of adiponectin are a risk factor for the progression to type 2 diabetes in the Japanese population: the Funagata study. Diabetes Care 26, 20152020.
Diez JJ and Iglesias P (2003) The role of the novel adipocyte-derived hormone adiponectin in human disease. Eur J Endocrinol 148, 293300.[Medline]
Filippi E, Sentinelli F, Trishitta V, Romeo S, Area M, Leonetti F, Di Mario U and Baroni MG (2004) Association of the human adiponectin gene and insulin resistance. Eur J Hum Genet, in press.
Goldfine AB and Kahn RC (2003) Adiponectin: linking the fat cell to insulin sensitivity. Lancet 352, 1431.
Goodarzi MO and Korenman SG (2003) The importance of insulin resistance in polycystic ovary syndrome. Fertil Steril 80, 255258.[Medline]
Hara K, Boutin P, Mori Y, Tobe K, Dina C, Yasuda K, Yamauchi T, Otabe S, Okada T, Eto K et al. (2002) Genetic variation in the gene encoding adiponectin is associated with an increased risk of type 2 diabetes in the Japanese population. Diabetes 51, 536540.
Hu E, Liang P and Spiegelman BM (1996) AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271, 1069710703.
Kissebah AH, Sonnenberg GE, Myklebust J, Goldstein M, Broman K, James RG, Marks JA, Krakower GR, Jacob HJ, Weber J et al. (2000) Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome. Proc Natl Acad Sci USA 97, 1447814483.
Lindsay RS, Funahashi T, Hanson RL, Matsuzawa Y, Tanaka S, Tataranni PA, Knowler WC and Krakoff J (2002) Adiponectin and development of type 2 diabetes in the Pima Indian population. Lancet 360, 5758.[CrossRef][Medline]
Lobo RA (2003) What are the key features of importance in polycystic ovary syndrome? Fertil Steril 80, 259261.[Medline]
Lord MJ, Flight IK and Norman RJ (2003) Metformin in polycystic syndrome: systematic review and meta-analysis. BMJ 327, 951956.
Matzuzawa Y, Funahashi T and Nakamura T (1999) Molecular mechanism of metabolic syndrome X: contribution of adipocytokines, adipocyte-derived bioactive substances. Ann N Y Acad Sci 892, 146154.
Menzaghi C, Ercolino T, Di Paola R, Berg AH, Warram JH, Scherer PE, Trischitta V and Doria A (2002) A haplotype at the adiponectin locus is associated with obesity and other features of the insulin resistance syndrome. Diabetes 51, 23062312.
Nishizawa H, Shimomura I, Kishida K, Maeda A, Kuriyama H, Nagaretani H, Matsuda M, Kondo H, Furuyama N, Kihara S et al. (2002) Androgens decrease plasma adiponectin, an insulin-sensitizing adipocyte derived protein. Diabetes 51, 27342741.
Okamoto Y, Arita Y, Nishida M, Muragushi M, Ouchi N, Takahashi M, Igura T, Inui Y, Kihara S, Nakamura T et al. (2000) An adipocyte-derived plasma protein, adiponectin, adheres to injured vascular walls. Horm Metab Res 32, 4750.[Medline]
Orio F Jr, Palomba S, Cascella T, Milan G, Mioni R, Pagano C, Zullo F, Colao A, Lombardi G and Vettor R (2003) Adiponectin levels in women with polycystic ovary sindrome. J Clin Endocrinol Metab 88, 26192623.
Ouchi N, Kihara S, Arita Y, Maeda K, Kuriyama H, Okamoto Y, Hotta K, Nishida M, Takahashi M, Nakamura T et al. (1999) Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin. Circulation 100, 24732476.
Ouchi N, Kihara S, Arita Y, Nishida M, Matsuyama A, Okamoto Y, Ishigami M, Kuriyama H, Kishida K, Nishizawa H et al. (2001) Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages. Circulation 103, 10571063.
Panidis D, Kourtis A, Farmakiotis D, Mouslech T, Rousso D and Koliakos G (2003) Serum adiponectin levels in women with polycystic ovary syndrome. Hum Reprod 18, 17901796.
Panidis D, Farmakiotis D, Kourtis A and Rousso D (2004a) Resistin as a local factor in polycystic ovary syndrome: a novel view of adipocytokines? Hum Reprod, in press.
Panidis D, Koliakos G, Kourtis A, Farmakiotis D, Mouslech T and Rousso D (2004b) Serum resistin levels in women with polycystic ovary syndrome. Fertil Steril 81, 361366.[CrossRef][Medline]
Reusch JE (2002) Current concepts in insulin resistance, type 2 diabetes mellitus, and the metabolic syndrome. Am J Cardiol 90, 19G26G.[Medline]
Sam S and Dunaif A (2003) Polycystic ovary syndrome: syndrome XX? Trends Endocrinol Metab 14, 365370.[CrossRef][Medline]
Schaffler A, Barth N, Palitzsch KD, Drobnik W, Scholmerich J and Schmitz G (2000) Mutation analysis of the human adipocyte-specific apM-1 gene. Eur J Clin Invest 30, 879887.[CrossRef][Medline]
Scherer PE, Williams S, Fogliano M, Baldini G and Lodish HF (1995) A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem 270, 2674626749.
Seow KM, Juan CC, Wu LY, Hsu YP, Yang WM, Tsai YL, Hwang JL and Ho LT (2004) Serum and adipocyte resistin in polycystic ovary syndrome with insulin resistance. Hum Reprod 19, 4853.
Spranger J, Kroke A, Mohlig M, Bergmann MM, Ritsow M, Boeing H and Pfeiffer AFH (2003) Adiponectin and protection against type 2 diabetes mellitus. Lancet 361, 226228.[CrossRef][Medline]
Stumvoll M, Tschritter O, Fritsche A, Staiger H, Renn W, Weisser M, Machicao F and Haaring H (2002) Association of the T-G polymorphism in adiponectin gene (exon 2) with obesity and insulin sensitivity: interaction with family history of type 2 diabetes. Diabetes 51, 3741.
Takahashi M, Arita Y, Yamagata K, Matsukawa Y, Okutomi K, Horie M, Himomura I, Hotta K, Kuriyama H, Kihara S et al. (2000) Genomic structure and mutations in adipose-specific gene, adiponectin. Int J Obes Relat Metab Disord 24, 861868.[CrossRef][Medline]
The Rotterdam ESHRE/ASRM Sponsored PCOS Consensus Workshop Group (2004) Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 19, 4147.
Tschritter O, Fritsche A, Thamer C, Haap M, Shirkavand F, Rahe S, Staiger H, Maerker E, Haring H and Stumvoll M (2003) Plasma adiponectin concentrations predict insulin sensitivity of both glucose and lipid metabolism. Diabetes 52, 239243.
Ukkola O, Ravussin E, Jacobson P, Sjostrom L and Bouchard C (2003) Mutations in the adiponectin gene in lean and obese subjects from the Swedish obese subjects cohort. Metabolism 52, 881884.[CrossRef][Medline]
Vionnet N, Hani E, Dupont S, Gallina S, Francke S, Dotte S, De Matos F, Durand E, Lepretre F, Lecoeur C et al. (2000) Genomewide search for type 2 diabetes-susceptibility genes in French whites: evidence for a novel susceptibility locus for early onset diabetes on chromosome 3q27-qter and independent replication of a type 2-diabetes locus on chromosome 1q21-q24. Am J Hum Genet 67, 14701480.[CrossRef][Medline]
Yang WS, Hsiung CA, Ho LT, Chen YT, He CT, Curb JD, Grove J, Quertermous T, Chen YDI, Kuo SS et al. (2003a) Genetic epistasis of adiponectin and PPAR2 genotypes in modulation of insulin sensitivity: a family-based association study. Diabetologia 46, 977983.[CrossRef][Medline]
Yang WS, Tsou PL, Lee WJ, Tseng DL, Chen CL, Peng CC, Lee KC, Chen MJ, Huang CJ, Tai TY et al. (2003b) Allele-specific differential expression of a common adiponectin gene polymorphism related to obesity. J Mol Med 81, 428434.[CrossRef][Medline]
Zawadski JK and Dunaif A (1992) Diagnostic criteria for polycystic ovary syndrome: towards a rational approach. In Dunaif A, Givens JR, Haseltine FP, and Merriam GE (eds) Polycystic Ovary Syndrome. Current Issues in Endocrinology and Metabolism. Blackwell Scientific Publications, Boston, MA, USA, pp. 377, Hershman, S.M (series ed.).
Zietz B, Barth N, Scholmerich J, Schmitz G and Schaffler A (2001) Gly15Gly polymorphism within the human adipocyte specific apM-1 gene but not Tyr111His polymorphism is associated with higher levels of cholesterol and LDL-cholesterol in caucasian patients with type 2 diabetes. Exp Clin Endocrinol Diabetes 109, 320325.[CrossRef][Medline]
Submitted on January 26, 2004; accepted on April 30, 2004.