Molecular & genetic factors contributing to insulin resistance in polycystic ovary syndrome.
Polycystic ovary syndrome (PCOS) is the most common endocrine
disorder of unknown etiology. Insulin resistance is very common and
plays a central pathogenic role in PCOS. During last decade several
studies have been conducted to understand the mechanisms contributing to
the state of insulin resistance and insulin-induced hyperandrogenemia in
PCOS. Insulin signaling pathways have been dissected in different
insulin responsive tissues such as skeletal muscles, adipose tissues,
fibroblasts as well as ovaries to elucidate the mechanism. These studies
suggest a post receptor signaling defect where metabolic action of
insulin is affected but not the steroidogenic and mitogenic actions.
Despite advancement in these studies gaps exist in our understanding of
the mechanism of insulin resistance as well as insulin-induced
steroidogenesis in PCOS. The syndrome is now considered as a complex
multigenic disorder. Efforts are ongoing to dissect the variants of
genes from multiple logical pathways which are involved in
pathophysiology of the syndrome. But still today no gene has been
emerged as universally accepted susceptibility gene for PCOS. This
review briefly describes the lacunae along with the current status of
molecular events underlying insulin resistance and the contribution of
insulin signaling pathway genes in pathogenesis of PCOS along with
future researchable areas.
Key words Hyperandrogenemia--insulin signaling--PCOS--steroidogenesis--phosphorylation
(Development and progression)
Stein-Leventhal syndrome (Risk factors)
|Publication:||Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2010 Indian Council of Medical Research ISSN: 0971-5916|
|Issue:||Date: June, 2010 Source Volume: 131 Source Issue: 6|
|Geographic:||Geographic Scope: India Geographic Code: 9INDI India|
Polycysticovary syndrome (PCOS) is a complex and heterogeneous disorder, affecting approximately 7 per cent of women in reproductive age (1). It is characterized by chronic anovulation, hyperandrogenemia, altered LH: FSH ratio (>2/3:1) and polycystic ovaries. The syndrome is a major cause of anovulatory infertility (2). The aetiopathogenesis of this syndrome still remains elusive but likely to be mutifactorial consisting of genetic and environmental components.
Insulin resistance (IR) is now known to be intrinsic to this disorder, present in approximately 50-70 per cent of these women independent of obesity, and contributing in a major way to its pathogenesis (2,3). Women with PCOS are frequently obese which contributes an extrinsic component of IR. It is known that IR progresses towards the development of compensatory hyperinsulinemia, which drives hyperandrogenemia in these women (4). Excess androgen levels lead to menstrual disturbances, development of ovarian cysts, hirsutism and other related disorders. IR also increases the risk for development of glucose intolerance, type 2 diabetes mellitus (T2DM), hypertension, dyslipidaemia and cardiovascular abnormalities in these women (3,5) (Fig. 1).
[FIGURE 1 OMITTED]
Hyperinsulinemia and hyperandrogenemia are thus two principal features of PCOS and their cause and effect relationship is still debated (2,6). However, several evidences suggest hyperinsulinemia to be the primary factor contributing to the ovarian hyperandrogenemia. Pharmacological reduction of insulin levels has been found to improve hyperinsulinemia as well as hyperandrogenemia and restore ovulation in the women with PCOS (2,4,6). However, reduction of androgen levels by bilateral oophorectomy or administration of GnRH agonist (6) or antiandrogenic compounds (2) were seen to have no effect on IR or hyperinsulinemia in the PCOS women which would have been expected, if hyperandrogenemia was the cause of hyperinsulinemia.
In PCOS the "central paradox" is that ovary remains sensitive to insulin action to produce androgens in spite of systemic insulin resistant state; whereas classical target organs of insulin as well as ovary remain resistant to its metabolic activity. The number and affinity of insulin receptors have been found to be optimal in different insulin target tissues and ovary, and also no structural and mutational abnormalities could be detected in the PCOS women (2,4,9,10). Thus a post-receptor binding defect in the insulin signaling pathway appears to play an important role in the etiology of selective IR. Although several in vitro and in vivo studies have been carried out in various tissues (adipocytes, fibroblasts, myocytes and ovarian cells) (2,4,11-28) to elucidate the potential mechanism of IR and insulin derived hyperandrogenemia (ovarian cells) (1,24-28) in PCOS women, the data is not yet conclusive. This review briefly describes the current understanding of the mechanism of IR and insulin induced hyperandrogenemia in PCOS, emphasizing the need for future research.
Signaling pathways of insulin action: A brief overview
Insulin exerts a wide range of pleiotropic actions at the target tissue such as cellular metabolism, growth and differentiation, via different signaling pathways. Binding of insulin to extracellular [alpha] subunits of insulin receptor (INSR) leads to activation of intrinsic tyrosine kinase and autophosphorylation of its [beta] subunits. Activated INSR phosphorylates a number of substrates like the insulin receptor substrate family (IRS 1-4), Gab-1, Cbl, APS and Shc isoforms, and signal regulatory protein (SIRP) family members which bind to INSR (29). Phosphorylated IRS proteins act as docking sites for several intracellular proteins such as Grb2, NcK and the regulatory subunit p85 of phosphatidylinositol 3-Kinase (PI3K), which mediate different actions of insulin. PI3K activation is crucial for metabolic actions, such as GLUT4 translocation, glucose transport, glycogen synthesis and protein synthesis, however the downstream signaling proteins of PI3K pathway is still not clear, probably it activates Akt and atypical PKC isoforms [lambda] and [zeta]. A substrate of Akt, AS160 is involved in GLUT4 translocation from intracellular vesicles to the plasma membrane, which results in rapid entry of glucose into the cell (22). Another pathway leading to GLUT4 translocation involves the insulin receptor-mediated phosphorylation of the scaffolding protein CAP (c-Cbl Associated Protein) and formation of the CAP:Cbl:CrkII complex. This complex, through its interaction with flotillin, localizes to lipid rafts facilitating GLUT4 translocation (30). The mitogenic action is mediated through binding of phosphorylated IRS1/2 or Shc with Grb-2/SOS complex leading to p21Ras and Raf-1 activation of mitogen-activated protein kinase pathway (MAPK) (21). PI3K probably facilitates the mitogenic response as well (Fig. 2).
Studies with ovarian tissues
Increased androgen production by ovarian cells is the classical endocrine phenotype of PCOS (11,31). The genomic and molecular studies demonstrated multiple alterations in the steroidogenic machinery of theca cells from PCOS women viz., overexpression of various proteins including luteinizing hormone (LH) receptor, INSR, lipoprotein receptor [high-density lipoprotein (HDL) and low-density lipoprotein (LDL)], steroidogenic acute regulatory protein (StAR), [P.sub.4]50 side-chain cleavage ([P.sub.4]50scc), 3[beta]-hydroxysteroid dehydrogenase (3[beta]-HSD) and cytochrome [P.sub.4]50c17 (CYP-17) (1,11). All these contribute to excess production of progesterone ([P.sub.4]), 17[alpha]-hydroxyprogesterone and testosterone as compared to normal theca cells (1,11). The role of insulin in ovarian function becomes evident from the observations of severe ovarian hyperandrogenemia in women with syndromes of extreme insulin resistance (4). Further, identification of INSR throughout the ovary and the ability of insulin to stimulate biosynthesis of androgens, estrogen and [P.sub.4] in ovarian cell culture suggest that ovary is another target organ of insulin action (4,24). These findings clearly indicate that insulin may play a role in normal follicular development and hence in a variety of insulin resistant states ovarian dysfunctions are manifested (4).
[FIGURE 2 OMITTED]
The molecular mechanism by which insulin augments androgen synthesis in ovary of PCOS women, in the systemic insulin resistant state, is still elusive. However, to explain this 'paradox' several hypotheses have been proposed (4). The first one suggests that the steriodogenic action of insulin might be mediated via IGF-1 receptor or hybrid insulin/IGF-1 receptor. However, insulin can act through IGF-1 receptor only when its circulating levels are extraordinarily high but in PCOS women it is only moderately high. Evidence indicates that insulin stimulates steriodogenesis in granulosa cells (GCs) and theca cells from both normal and PCOS ovaries via its cognate receptor (4). The second hypothesis proposes that the signaling cascade for metabolic action of insulin in ovary becomes divergent from the one, which mediates steroidogenic action after binding of insulin to INSR. Insulin may activate steroidogenesis via several pathways, either via cross talk with LH-induced cAMP accumulation, which in turn might activate PI3K activity or MAPK pathway or via alternate pathways of insulin signaling. Primarily, steroidogenesis in theca cells is regulated by LH, which acts via cAMP dependent protein kinase pathway (PKA) (11). Insulin alone was not found to increase cAMP but it enhanced LH-induced cAMP accumulation in porcine theca cells (25). LH and insulin independently and also synergistically could increase expression of genes in androgen biosynthesis pathway viz., StAR and CYP17, and also augment androstenedione and [P.sub.4] biosynthesis in primary culture of porcine theca cells (25). A stable cAMP analog (8-BrcAMP) could mimic the same effect on steroidogenesis, StAR and CPY17 mRNA expression but when it was co-administered with insulin it could enhance only [P.sub.4] synthesis and StAR gene expression but not CPY17 expression and androstenedione biosynthesis (25). This indicated the mechanism involved in the expression of CPY17 is not perhaps cAMP dependent or may involve steps downstream of cAMP accumulation. On the other hand, study with cultured human theca cells showed insulin alone had no effect on 17[alpha]-hydroxylase activity or CPY17 expression but requires concomitant activation of cAMP signaling pathway by forskolin (27). This different effect of exogenous or endogenous cAMP along with insulin on CPY17 gene expression indicates the possibility of different cellular response mediated by exogenous or endogenous cAMP. The stimulation of 17[alpha]-hydroxylase activity was blocked by PI3K inhibitor but not by MAPK kinase (MEK) inhibitor, indicating insulin action on 17[alpha]-hydroxylase activity is mediated via PI3K pathway not via MAPK pathway (27). However, this response of insulin is different from its effect on glucose metabolism which do not require co-activation of cAMP pathway, again emphasizing the divergence of signaling pathways distal to PI3K for metabolic and steroidogenic action (28). However, both MEK1/2 and ERK1/2 phosphorylation were markedly reduced and were associated with increased CYP17 mRNA abundance and androgen production in theca cells from PCOS women compared to cells from normal women (28). Besides, MEK1/2 activity was reduced and CYP17 mRNA abundance and dehydroepiandrosterone (DHEA) activity was increased even in absence of insulin treatment in PCOS cells suggesting that alteration in MAPK pathway may plays a role in pathogenesis of excess androgen biosynthesis via an insulin independent mechanism (28).
Insulin alone or with FSH can augment [P.sub.4]50 aromatase activity in human GCs (32) and can stimulate oestradiol ([E.sub.2]) production by GCs isolated from polycystic ovary (PCO) (33). Aromatase activity has been reported to be low in GC from PCOS ovary (33). On the contrary a significant increase in basal aromatase activity had been reported in GCs isolated from PCO ovary and these GCs were more sensitive and hyperresponsive not only to FSH but also to insulin and IGF-1 (34). In these studies the stimulatory effect of insulin on aromatase activity was observed only at supraphysiological dose. Insulin even at physiological concentration could enhance [E.sub.2] and [P.sub.4] production by GCs from both PCOS and normal ovaries24. Interestingly, insulin along with FSH augmented [P.sub.4] production by GCs from normal and PCOS ovary but enhanced [E.sub.2] secretion only by GCs from anovulatory PCOS women and not from normoovulatory PCOS and normal women (24). As anovulatory PCOS women are known to be more insulin resistant than normoovulatory PCOS, these suggest that the steroidogenic machinery may be more responsive to insulin in a systemic insulin resistant state. Recent data also showed that insulin can regulate the expression as well as activity of aromatase and also the expression of 3[beta]-HSD in human luteinized GCs (35). Experimental data from cultured human GCs from both control and PCOS women suggest that neither PI3K nor MAPK pathway are involved in insulin induced steriodogenesis (36) which is in contrast to theca cells where PI3K has been shown to be involved (27). Cholesterol which is the substrate for steroid biosynthesis is transported into ovarian cell via LDL receptor. Sekar and Veldhuis (26) demonstrated insulin and LH synergistically upregulated transcription of LDL receptor in porcine GCs via mechanism that involves PKA, PI3K and MAPK pathways. Insulin treatment elevates the [P.sub.4]50ssc mRNA levels in bovine luteal cell (37). Insulin when co-administered with LH the expression of [P.sub.4]50ssc mRNA was much higher that that occurred only with LH26. All these data support that elevated insulin levels can affect steroid production in human GCs and alter the menstrual cycle and fertility. Though steriodogenic action of insulin is maintained, the metabolic action is affected in PCOS women. A decrease in lactate production but not [P.sub.4] secretion by granulosa-lutein cells, isolated from PCOS women have been reported (38) (Table I).
Another hypothesis proposed to explain the 'paradox' is serine phoshorylation theory, based on the observation that serine phosphorylation of the main regulatory enzyme of androgen biosynthesis i.e., [P.sub.4]50c17 appears to modulate its 17, 20 lyase activity and subsequent androgen production (6). In a subgroup of PCOS women, IR appears to be related to excess serine phosphorylation of the [beta] subunit of INSR (2). The mechanism of serine phosphorylation is still unknown but evidence suggests it may be due to serine/threonione kinase extrinsic to INSR or due to an inhibitor of a serine/threonine phosphatase (19). This leads to the postulation that a single hypothetical kinase might phosphorylate both INSR and [P.sub.4]50c17 and thus account for both hyperandogenemia and hyperinsulinemia in a subgroup of PCOS women (6).
Identification of the kinase or the regulatory factors responsible for the serine phosphorylation is required to prove this hypothesis.
Recently it has been debated whether PCOS is a state of ovarian hypersensitivity to insulin or a state of preserved ovarian sensitivity inspite of systemic IR. Baillargeon and Nestler (39) postulated the former view as the cultured theca cells from PCOS women secrete more androgen upon insulin stimulation compared to normal cells. Furthermore, normoinsulinemic PCOS women treated with insulin sensitizing drugs exhibit substantial reduction in ovarian androgen production though the reduction in insulin level is modest, suggesting increased insulin sensitivity of the androgenic pathway. Conversely, Poretsky (40) believes ovarian sensitivity is maintained in PCOS women inspite of systemic IR, as ovarian cells from PCOS women secrete higher amount of androgens not only in response to insulin but regardless of the stimulus used. He emphasized that the signaling pathways mediating ovarian effects of insulin other than glucose transport are different from classical insulin signaling pathways. Further studies are required to delineate the alternate signaling pathway mediating ovarian effect of insulin, their interaction with IGF-1 pathway or any other pathways that are hypersensitive to physiological concentrations of insulin in PCOS.
In recent years, PCOS research has focused on comparative gene expression profiles of different components of ovarian tissue such as theca cells, ovary and oocytes of PCOS and normal women (41-44),. These studies have provided some leads about the expression profile of insulin signaling and functional pathway related genes and their possible transcriptional regulation. The first study by Wood et al (41) on theca cells from PCOS and normal ovary showed differential expression of 346 genes. To understand the transcriptional regulation of these genes, the promoter regions were analyzed by in silico approach in our lab (45). The analysis identified four putative transcription factor binding sites (TFBS) i.e., Staf, E47, CCAAT Box and CRE-BP in the promoter regions of co-expressed genes which were overexpressed in the PCOS theca cells. Members of CCAAT box family are known to be associated with CYP11A1, CYP17 and 3[beta]HSD as well as genes related to their action such as GLUT4 and peroxisome proliferator activated receptor-gamma (PPAR-[gamma]). Multiple transcriptionally active CRE-BP sites have been found in human insulin gene promoters. E47 plays an important role in the regulation of insulin transcription in the pancreatic [beta]-cells and thus in maintenance of insulin sensitivity (45). Another in vitro gene expression study with normal and PCOS ovaries revealed altered expression of several genes involved in insulin function like PDK4 (pyruvate dehydrogenase kinase 4), PIGH (phosphatidylinositolglycan class H) and UDP-GalNAcPP (UDP-GalNAc pyropho sphorylase) and some genes involved in insulin mitogenic pathway, mainly those in MAPK pathway, such as RPS6KA2 (44). PDK4 has recently been shown to play a role in the pathogenesis of insulin resistance in T2DM (44). Similar studies with oocyte revealed the presence of putative binding sites for androgen receptor (AR), PPAR-[gamma], PPAR-[gamma]/Retinoic X Receptor (RXR) in the promoter regions of many genes differentially expressed in PCOS. PPAR-[gamma] is a ligand-activated transcription factor involved in glucose and lipid metabolism are known to modulate insulin signaling pathway. Over expression of PPAR-[gamma] gene in PCOS ovary (43) and the presence of PPAR-[gamma] binding sites in differentially expressed genes suggest its role in pathogenesis of the syndrome but the mechanism needs to be elucidated. It has been demonstrated that insulin can phosphorylate and activate PPAR-[gamma] in absence of endogenous PPAR-[gamma] ligand (42). Thiazolidinidiones (TZDs), the synthetic PPAR-[gamma] agonist can restore insulin sensitivity and ameliorate hyperandrogenemia in PCOS women4. TZDs have been shown to exert direct effect on ovarian androgen production through both insulin independent and insulin sensitizing effect. They directly enhance [P.sub.4] and IGFBP-1 synthesis and inhibit [E.sub.2] and testosterone production and also reduce insulin induced testosterone production (46). The indirect effect of TZDs is mediated by its systemic insulin-sensitizing action through reduction in insulin level. Insulin and TZDs independently have been shown to increase expression of INSR, IRS-1, PPAR[gamma] and StAR protein in human ovarian cells (46). This indeed indicates a crosstalk between insulin signaling, PPAR[gamma] and steriodogenic pathways which may constitute a novel regulatory mechanism in ovarian function. The effect of insulin on steroidogenesis machinery in ovarian cell is summarized in Table 1. Further molecular studies are needed to dissect the role of these genes and their downstream pathways in the pathophysiology of this syndrome.
Studies with adipocytes
Both lean and obese women with PCOS exhibit significant decrease in maximal insulin-stimulated glucose utilization in vivo and in isolated adipocytes, suggesting an intrinsic abnormality, independent of obesity (2). Ciaraldi et al (12) observed reduced insulin sensitivity but normal insulin responsiveness in adipocytes isolated from PCOS women with IR. The receptor kinase activity was optimal inspite of decrease in insulin induced INSR autophosphorylation suggesting a defect in signaling cascade between INSR and glucose transport (12). On the contrary, Lystedt et al (13) reported no differences in the insulin sensitivity to glucose uptake in cultured adipocytes of PCOS women compared to controls. This discrepancy might be result of the fact that former group12 studied fat cells immediately after surgery, when the cells were in the state of IR due to surgical stress whereas, the later group (13) allowed adipocytes to recover before initiating the study. The expression of IRS-1, Akt1/2, PKC[zeta] as well as their phosphorylation status and PI3K activity were similar in adipocytes isolated from both control and PCOS women (12). On the contrary a reduction in IRS-1 expression as well as its tyrosine phosphorylation has been reported (14,15). The expression of IRS-2 was normal but phosphorylation was decreased in adipocytes from insulin resistant PCOS group compared to noninsulin resistant PCOS women and controls (16). Rosenbaum's group (20) observed GLUT4 expression, was significantly reduced in adipocytes from PCOS women, independent of obesity and correlated well with diminished insulin responsiveness, but Ciraldi et al12 found no alteration in expression. The expression of CAP or cbl protein was not different in adipocytes from control and PCOS (12). A recent study with subcutaneous preadipocytes of PCOS women concluded that there is no intrinsic defect of insulin signaling in adipose cell lineage, the IR is due to the factors present in the in vivo environment (18).
Visceral adiposity is the commonest phenotype seen in both lean and obese PCOS, which releases increased amounts of free fatty acids (FFA), known to plays a pathophysiological role in IR directly by affecting insulin signaling. Catecholamine-activated lipolysis was shown to be reduced in subcutaneous (SC) adipocytes (47), but enhanced in visceral fat (48) of PCOS women which may be due to altered expression of lipolysis regulatory proteins in these two fat depots of controls and PCOS women. This partly explains the metabolic defect observed in PCOS subjects, where lipolysis resistant SC fat cells promotes obesity and enhanced lipolysis in visceral fat increases FFA levels.
Adipose tissue factors in pathogenesis of PCOS
It is now well recognized that adipose tissue functions as a highly specialized endocrine and paracrine organ producing an array of adipokines like adiponectin, leptin, resistin, TNF-[alpha] etc. which affect insulin sensitivity. The relationship of these adipokines with IR and obesity in PCOS women are conflicting.
Adiponectin, a key adipokine in mediating relationship between body weight and insulin sensitivity, protects against IR and T2DM (49). Several studies reported lower serum adiponectin levels in PCOS women compared to controls (49-52) and some of them have observed no significant difference (53). Recent findings indicate that the effect of adiponectin on insulin sensitivity is mediated primarily by high molecular weight (HMW) form of adiponectin, but studies showed no difference in HMW adiponectin in PCOS and controls (53). A recent metanalysis revealed lower adiponectin levels in PCOS women which showed association with IR but not with total testosterone levels (53).
Resistin, another adipokine, which is significantly increased in insulin-resistant mice and genetic or diet-induced obese mice has been shown to be down regulated by insulin sensitizing agents (54), suggest to mediate a link between obesity and IR in mice, but this relationship remains controversial in human. Circulating resistin level is proportional to degree of adiposity and decreased adiponectin and increased resistin have been linked to the development of IR (50). Most of the studies have not found any difference in resistin levels between PCOS and controls (50,52) however, increased expression of resistin mRNA55 in adipocytes as well as elevated serum resistin levels was observed in PCOS women (55,56).
Leptin, is a key hormone in energy homeostasis and neuroendocrine function, may involve in pathogenesis of hyperandrogenism and infertility in women with PCOS (57). High leptin levels have been deemed as a component of the metabolic syndrome, obesity, IR, hypertension and dyslipidemia (58), and hyperleptinemia may lead to leptin resistance. Some studies showed similar leptin levels in both PCOS women and weight matched controls but showed significant association between leptin levels and BMI (50,59), and others reported high circulating levels of leptin in the PCOS women (49,59).
Retinol-binding protein 4 (RB[P.sub.4]) has been implicated in IR and reported to be high in insulin resistant humans with obesity and T2DM (51). Elevated levels of serum RB[P.sub.4] as well as increased mRNA and protein expression in adipose tissue were reported in overweight PCOS women (60). On the other hand, similar serum levels of RB[P.sub.4] were observed in PCOS and controls but its relationship with IR is contradictory (51,61). Barber et al (51) clearly demonstrated that RB[P.sub.4] levels correlate with visceral fat depot. Recently, Diamanti-Kandarakis et al (62) observed lower levels of both RB[P.sub.4] and free plasma RB[P.sub.4] (a more sensitive marker of circulating RB[P.sub.4]) in insulin-resistant PCOS subjects than controls, suggesting lack of relationship between IR and RB[P.sub.4]. Aigner et al (63) reported a positive correlation between RB[P.sub.4] and androgen levels and also clinical hirsutism scores in women with PCOS, suggesting a role in steroid metabolism, which needs further investigation. The serum levels of visfatin, another adipokine and its mRNA and protein expression in adipocytes were reported to be increased in PCOS women (64,65).
A gene expression study carried out by Corton et al (66) with omental adipose tissue from PCOS women showed overexpression of ectoenzyme nucleotide pyrophosphate phosphodiesterase 1 (ENPPI), which is a negative regulator of INSR tyrosine kinase activity and the regulatory p85[alpha] subunit of PI3K which resulted in decreased PI3K activity. Besides, several actin and myosin isoforms were downregulated which may result in reduced glucose transport. The upregulation of a serine/threonine kinase PKN2 was observed which may be involved in excess serine phosphorylation of different proteins in insulin signaling pathway as well as steroidogenic enzymes leading to impaired insulin action and enhanced steriodogenesis in PCOS.
Studies with fibroblasts
Increased basal autophosphorylation of serine residues of INSR and subsequent decrease in insulin stimulated tyrosine autophosphorylation of INSR were reported in 50 per cent of cultured skin fibroblasts from women with PCOS (19). Similar defect was also observed in vivo, indicating that the defect might be intrinsic. Excess serine phosphorylated INSR showed reduction in tyrosine phosphorylation of an artificial substrate, which again provides evidence of association between increased serine phosphorylation and decreased tyrosine kinase activity (67). On the other hand, the remaining 50 per cent of PCOS women in whom INSR autophosphorylation was normal, had similar degree of IR, which clearly indicate a signaling defect downstream of INSR. Fibroblast from PCOS women showed similar mitogenic action like other tissues but glycogen synthesis was affected (10). Optimal expression of IRS-1 and its associated PI3K activity in cultured fibroblasts of these women suggests that metabolic signaling defect lay either on a different signaling pathway or at steps that are downstream of IRS-1 mediated PI3K activation (10). On the contrary, Ciaraldi et al (20) found no difference in both metabolic and mitogenic actions of insulin in fibroblast culture from obese PCOS women, though adipocytes of same women demonstrated impaired glucose transport suggesting that the insulin signaling defect is possibly tissue specific (20). In cultured fibroblast, isolated from PCOS women, though Akt phosphorylation was normal, a decreased phosphorylation of GSK-3 was observed which led to decreased activation of glycogen synthase and diminished glycogen synthesis 21.
Studies with skeletal muscle
Skeletal muscle is the major site for insulin mediated glucose uptake. In vivo studies, where muscle biopsies were serially taken at different time points during euglycemic hyperinsulinemic glucose clamp, revealed normal steady state insulin levels but reduced insulin mediated glucose uptake (IMGU) in PCOS women along with decrease in insulin mediated IRS-1 associated PI3K activity (19). The expression of signaling proteins upstream of PI3K like INSR, IRS-1 and also p85 subunit of PI3K were normal but that of IRS-2 was markedly increased19,12. However, as IMGU was still decreased in PCOS which indicate that increased IRS-2 associated PI3K activity could not completely compensate for the defect. In muscle biopsies from PCOS women, impaired IMGU were paralleled by reduced insulin induced phosphorylation of Akt at [Ser.sup.473], [Thr.sup.308] and AS160, despite normal basal and insulin stimulated P13K activity, which improved by treatment with TZDs (22). This suggests that impaired glucose metabolism in skeletal muscle of PCOS women is mediated via signaling defect at Akt and AS160 level (22). Further research on other insulin sensitive tissues is warranted to determine whether similar defects exist or not.
Like fibroblasts, skeletal muscle from women with PCOS also showed impaired insulin action along with constitutive serine phosphorylation and decreased tyrosine kinase activity of INSR in vitro (2). In contrast to in vivo studies, basal and IMGU was significantly high in the cultured myotubes from obese PCOS women (68). Expression of INSR P subunit, IRS-2, p85 of PI3K as well as basal and insulin induced tyrosine phosphorylation of INSR were similar to controls. The IRS-1 expression and its phosphorylation at [Ser.sup.312] were significantly increased in PCOS women which might have inhibited its tyrosine phosphorylation and downstream signaling (68). Though IRS-1 related PI3K activity was normal, when normalized for increased IRS-1 expression, it was significantly decreased. IRS-2 mediated insulin signaling was also impaired as evident by decreased basal IRS-2 associated PI3K activity and decreased binding of p85 to IRS-2 upon insulin stimulation (68). In skeletal muscle, inspite of intrinsic defects in insulin signaling increased IMGU was observed in vitro, which indicates that interaction with in vivo environmental factors are required to manifest IR (69). Expression of GLUT4 was normal but GLUT 1 was significantly increased in PCOS which positively correlated with increased basal glucose uptake in skeletal muscle (69). On the other hand, Ciraldi et al (12) observed that myotubes from PCOS women displayed reduced IMGU and normal insulin sensitivity. The expression of GLUT4, insulin signaling proteins and insulin induced phosphorylation of Akt in skeletal muscle and myotubes did not differ between PCOS and controls (12). In vivo and in vitro studies with skeletal muscle showed that the mitogenic signaling via MEK1/2 and ERK 1/2 is constitutively enhanced in PCOS women and possibly at the level of Raf-1 (68). Inhibition of MEK activity by specific inhibitor resulted in decreased phosphorylation of IRS-1 at [Ser.sup.312] and increased association of IRS-1 with p85 of PI3K in both control and PCOS. This suggests that constitutive activation of ERK1/2 or ERK1/2 regulated kinase may inhibit the association of IRS-1 with p85 via IRS-1 [Ser.sup.312] phosphorylation and thus lead to impaired metabolic action of insulin in skeletal muscle of PCOS women (68).
In the light of these studies it becomes evident that mechanism underlying insulin resistance in PCOS women is tissue specific and pathway selective involving various molecules of insulin signaling and related pathways and the results are still inconclusive. The molecular mechanisms of insulin resistance and insulin mediated steroidogenesis are still elusive. The studies conducted so far have been mainly focused on PI3K, MAPK pathways and data suggest that these pathways may not be the primary ones involved in steroidogenesis therefore, alternate pathways of insulin signaling also need to be explored in PCOS. As PCOS is a heterogenous disorder it is also possible that the different subgroups of PCOS women may exist, having defects at different levels of insulin signaling. Recent reports on altered expression profile of several steroidogenesis and insulin signaling pathways genes in PCOS creates a need of integrated approach to study their molecular interactions to unravel the underlying mechanism involved in PCOS pathogenesis.
Genetic variants involved in insulin resistance in PCOS
Evidence from twin and family based studies have demonstrated an increased prevalence of PCOS and its phenotypic features in the relatives of women with PCOS, suggesting genetic factors underlying the syndrome (70,71). However, the mode of inheritance is still not clear and recent studies indicate that the disorder could be a complex trait where several gene variants interact with each other and along with the environmental factors in the manifestation of the syndrome. Ethnic variations in the prevalence of IR, obesity and PCOS and also their association with different gene variants have also been observed. A series of linkage association studies carried out in women with PCOS from USA showed a strong linkage between susceptibility to the disease itself and a dinucleotide marker in chromosome 19p13.2 (72-74). Most of the genetic studies have focused on candidate gene approach, selecting genes from multiple logical signaling pathways, implicated in the pathogenesis of PCOS, such as genes involved in steroid hormone biosynthesis and metabolism, insulin signaling, gonadotropin action and its regulation, and proinflammatory genes (70-72, 75,122). We briefly discuss here the most important findings published to date regarding the molecular genetic mechanisms underlying the association of PCOS with IR (Table II).
Insulin: The pancreatic [beta]-cell dysfunction in PCOS women appears to have a genetic predisposition. The minisatellite variations (variable number of tandem repeats, VNTR) upstream of the insulin gene locus (INS) regulates insulin expression. Watherworth et al (77) reported strong linkage and association between INS VNTR and PCOS but only in the form of preferential transmission of class III allele from heterozygous father but not from mother to daughter with PCOS. However later studies failed to confirm any linkage of INS with PCOS and also any association between class III INS VNTR alleles with hyperandrogenemia (71,72). Recently Ferk et alls reported a significant association of class III INS VNTR alleles with PCOS suggesting that an interaction of obesity and III/III INS VNTR genotype increases risk for development of PCOS.
Insulin receptor: Available evidence suggests IR in PCOS could be due to post-binding defects in insulin signaling (2,11). Hence INSR, being an integral part of insulin signaling, has been explored as a potential candidate gene. Linkage analysis studies have found an association of PCOS with the microsatellite marker D19S884, located on chromosome 19p13.2 and relatively close (1cM) to INSR (72-74). However, this association has not been supported by a case control study carried out in Spanish and Italian women with PCOS (79). As the number and affinity of INSR is not altered in PCOS but its tyrosine phosphorylation status and subsequent signaling is affected it suggests that the defect may lie in the [beta] chain (2,12). Several polymorphisms have been identified in women with PCOS of which more frequent were at exon 17 which encodes the partial tyrosine kinase domain of INSR (71,76,80-84). Among these polymorphisms, a C/T SNP at His1058 in exon 17 have been reported in studies with Caucasian, Chinese and Korean women (80,81,84). In the first two studies the frequency of T allele was found to be significantly different between obese and lean PCOS women, which suggest an association of this SNP with lean PCOS women (80,81). Our study in Indian women also confirmed this association with PCOS in the lean rather than obese women along with significant association with indices of IR and hyperandrogenemia in the same subgroup (83). Our findings suggest that the genetic pathogenesis of IR in PCOS could be different in lean and obese women. A Korean study however failed to confirm this association, rather reported an association of a novel C/T SNP at +176477 with PCOS (84). Another novel T/C polymorphism at Cys1008 in exon 17 of INSR has been reported to be associated with PCOS and decreased insulin sensitivity (82). A recent meta-analysis has reported no significant association of His 1058 C/T polymorphism with PCOS (85).
Insulin receptor substrates: IRS proteins are critical for insulin mediated signal transduction in insulin target tissues. Two common polymorphisms in IRS particularly Gly972Arg in IRS-1 and Gly1057Asp in IRS-2 have been shown to influence the susceptibility to T2DM and are associated with phenotypic features of PCOS (71,76,86-90). Several studies have reported a higher frequency of Gly972Arg allele of IRS-1 in PCOS women (89,91,92) but other studies failed to confirm this association (86,88,93). In two of these studies, this variant showed association with increased fasting insulin (87,93) whereas the study by Ehrmann et al (86) showed no association with glucose, insulin and androgen levels. A recent study in Taiwanese women reported absence of Gly972Arg and another Ala513Pro polymorphism in IRS-1 though majority of women with PCOS were either insulin resistant or glucose intolerant, thus suggesting IRS-1 variants may not play a role in glucose dysmetabolism in their population (90). Gly1057Asp variant of IRS-2 was shown not to associated with PCOS (86,87,94) However, a gene dosage effect of Arg972, IRS-1 on fasting insulin and Asp1057 variant of IRS-2 on 2 h glucose and insulin levels were observed among PCOS women (87). Contradicting reports are also available linking wild type genotype with increased 2 h glucose levels, (86,88). Interestingly, PCOS women with both IRS variants were found to have a higher degree of IR compared to those with only one IRS-2 variant and wild type allele, indicating functional impact of these polymorphisms on IR component of PCOS (87). A recent Mendelian meta analysis confirmed association of IRS-1 Gly972Arg with PCOS, which support the overall data and indicates a role of IRS in IR component of PCOS (85).
Ectoenzyme nucleotide pyrophosphate phosphodiesterase (ENPP1): ENPP1 (also known as PC-1, plasma cell differentiation antigen 1) has been identified as a factor potentially contributes to IR by binding to INSR and affecting its signaling. It has been reported to be overexpressed in different insulin target tissues of insulin resistant subjects (95) and also in adipose tissue of PCOS women96. Recent linkage and association studies suggest ENPP1 to be a susceptibility gene for IR and related abnormalities (95). A functional missense polymorphism K121Q in exon 4 has been reported to be associated with PCOS in a study from Finland (97) but other three studies showed negative results (92,98,99).
Peroxisome proliferator activated receptor gamma (PPAR-[gamma]): Genetic association of PPAR-[gamma] with IR and PCOS has evoked considerable interest in recent years, following an understanding of its role in diabetes. A common Pro12Ala polymorphism in PPAR-[gamma] has shown to be associated with PCOS in Finish and Korean women (100, 101). But some other studies have failed to confirm this association (98,102-105). However reports are available on association of this polymorphism with insulin sensitivity (104,106-108) with decreased basal metabolic rate and hyperandrogenemia (109). Another PPAR-[gamma] polymorphism 1431C/T (His447His) in exon 6 showed association with PCOS (101) and also with increased BMI and higher serum leptin levels (102). On the contrary, Antoine et al (108) reported no association with PCOS but T allele showed association with decreased free and total testosterone levels and as well as decreased IR in control women. A variant in PPAR-coactivator-1 (Gly482Ser) showed no association with PCOS (103).
Calpain-10: Calpain-10, a cysteine protease, has been shown to be associated with IR and susceptibility to T2DM (71). The potential association between several nucleotide polymorphisms (SNPs) in the CAPN10 (UCSNPs -45, -44, -43, -19, and -63) and PCOS susceptibility have been investigated with contradictory results. Ehrmann et al (110) found a haplotype combination (genotype 112/121) associated with higher insulin levels in African-Americans and which increases 2-fold risk of PCOS susceptibility in both African-Americans and Caucasian women. In Korean population specific haplotype and diplotypes of CAPN10 significantly increases the risk of development of PCOS, and few others decrease the risk (111). Studies in Spanish women reported association of UCSNP-44 (112) or UCSNP-45 (113) with PCOS susceptibility and showed further association of specific haplotypes with phenotypes, but no association observed between UCSNP-44 and PCOS in women from UK (114). A recent, study with German women genotyped eight variants of CAPN10 and a significant association of UCSNP-19 ins/del and UCSNP-56 with PCOS (115) were observed. Their metaanalysis showed a significant association only with ins/ del-19 with PCOS and they emphasized that CAPN10 may be an interesting candidate gene for PCOS (115).
Paraoxonase 1: Paraoxonase 1 encoded by the PON1 gene, is a serum high-density lipoprotein (HDL) associated enzyme with antioxidant property. Oxidative stress plays a central role in the pathogenesis of IR and cardiovascular disease. Three genomic variants of PON1 have been studied in a Spanish population, which showed increased frequency of -108 C/T polymorphism in promoter region of PON1 in PCOS women, whereas other two Leu55Met, and Gln192Arg polymorphisms in coding region showed no association (98). However, subjects homozygous for Met55 alleles presented with a higher BMI and increased indices of IR (98).
Adiponectin: Most of the studies investigating the association of genetic variations in the adiponectin gene (ADIPOQ, T45G in exon 2 and G276T in intron 2) with pathogenesis of PCOS have provided negative results (52,98,116), but some of them showed relation with serum adiponectin levels (116,117). A study with German PCOS women showed a higher prevalence of homozygous G allele of T45G polymorphism in PCOS women than in controls, but this was not associated with insulin resistant phenotype (94). Another study in Chinese women showed association of both T45G and G276T polymorphisms with PCOS and carriers of polymorphic genotype at G276T had decreased levels of serum adiponectin (117). Recently a Japanese study failed to show any association with -11377 polymorphism in promoter region of ADIPOQ with PCOS (118).
Resistin: The resistin gene (RETN) maps to the region on chromosome 19 which harbors possible candidate gene for PCOS (72,73). Three groups investigated the possible association of promoter region polymorphism (-179C/G or -420C/G) in RETN with PCOS, its phenotypes and also with serum resistin levels with none reporting any association with development of PCOS (52,119,120). However Xita et al (120) observed that PCOS women with CC genotype of -179C/G SNP had increased BMI as compared to women with polymorphic genotype, indicating an association of this polymorphism with adiposity in PCOS. The other SNP -420C/G showed no association with subphenotypes of PCOS (IR and obesity) or with serum levels of resistin (52, 119) but another study with Japanese women showed association with PCOS (118).
Leptin and leptin receptor: Leptin (LEP) and leptin receptor (LEPR) are attractive candidate genes for PCOS. A study in Finish population has not found any variation in coding region of LEP in PCOS women121. Regarding LEPR no association between its polymorphisms (exon 2, 4, 12 and 3' UTR pentanucleotide insertion) and PCOS susceptibility were observed (121,122). However in this Finnish study, carriers of polymorphic genotype of exon 12 (Lys656Asn) and LEPR 3'UTR (insertion) showed low serum insulin levels compared to wild type, suggesting a possible role of LEPR in regulation of insulin levels (121).
From the above review it is clear that although a genetic basis of PCOS is univocally accepted inspite of having several positive results, till today no gene has emerged as universally accepted susceptibitily gene for PCOS. The conflicting results might be attributed in part due to lack of universally accepted diagnostic criteria, relatively small sample size, failure to replicate results in independent studies, inadequate number of gene and their variants analyzed, presence of clinical heterogeneity among PCOS and ethnic variation between populations. To overcome this, uniformity in diagnosis of PCOS and sub-classification of cases according to sub-phenotypes, improved application of the candidate gene approach by selecting genes from expression studies, using haplotype-based analysis, replicating results in large cohorts and genome-wide approaches to maximize the chance of identifying an association is needed.
Although a large body of research has been devoted to understand the mechanisms underlying IR and insulin induced hyperandrogenaemia in PCOS, the data are still inconclusive. Evidence suggests that there is a post-receptor divergence in the insulin signaling pathway where metabolic activity is affected but not its steroidogenic and mitogenic activity. Use of high throughput techniques like pathway specific genomic and proteomic approaches may help to identify novel factors associated with insulin action in the target tissues. Moreover, subphenotyping PCOS women according to their insulin resistant status, may provide further insights to understand the molecular mechanism of insulin resistance in PCOS and also to develop novel therapeutic approaches to overcome the defect. The upcoming metabolomic research approach needs to be adapted to elucidate the aetiological mechanism involved in PCOS. Most of the genetic studies so for have used candidate gene approach. As PCOS is a heterogeneous and complex disorder, studies to elucidate function of genes in isolation will not be of much relevance. A genome wide scan or SNP microarray with phenotypically defined subgroup of PCOS may help to elucidate the different molecular defects associated with different phenotypes. As PCOS is a multigenic disorder an integrated approach of functional genomics (gene variations, gene expressions, protein-protein interaction and phenotypic variations) is needed to understand the genotype-phenotype correlation and extend it to understand interaction between different genes in manifestation of the syndrome and also its underlying pathogenesis.
Received May 15, 2008
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Reprint requests: Dr Srabani Mukherjee, Department of Molecular Endocrinology, National Institute for Research in Reproductive Health (ICMR), Jehangir Merwanji Street, Parel, Mumbai 400 012, India e-mail: email@example.com, firstname.lastname@example.org
Srabani Mukherjee & Anurupa Maitra
Department of Molecular Endocrinology, National Institute for Research in Reproductive Health (ICMR) Mumbai, India
Table I. Insulin effect on steroidogenic machinery in ovary Targets Cell Effect Stimulant LDL Receptor GC [up arrow] Insulin +LH StAR mRNA Theca [up arrow] Insulin, LH, Insulin +LH CYP11A mRNA LGC [up arrow] Insulin [up arrow] Insulin CYP17 mRNA Theca [up arrow] Insulin, LH, Insulin + LH [up arrow] Insulin + Forskolin 17[alpha]-hydroxylase Theca [up arrow] Insulin + Forskolin activity [P.sub.4] secretion GC [up arrow] Insulin [up arrow] Insulin [P.sub.4]50 aromatase GC [up arrow] Insulin LGC [up arrow] Insulin 3[beta]-HSD mRNA LGC [up arrow] Insulin Targets Exerted via References LDL Receptor PKA, PI3K, MAPK Sekar et al (16) StAR mRNA via cAMP Zhang et al (25) CYP11A mRNA N.d Mamluk et al (37) N.d Sekar et al (26) CYP17 mRNA Not completely cAMP Zhang et al (25) dependent cAMP Munir et al (27) 17[alpha]-hydroxylase PI3K Munir et al (27) activity [P.sub.4] secretion Not via PI3K and MAPK Poretsky et al (36) N.d Willis et al (24) Rice et al (38) [P.sub.4]50 aromatase N.d Pierro et al (34) N.d Fedorcsak et al (35) 3[beta]-HSD mRNA N.d Fedorcsak et al (35) LDL-R, low density lipoprotein receptor; StAR, steroidogenic acute regulatory protein; 3[beta]-HSD, 3[beta]-hydroxysteroid dehydrogenase; [P.sub.4], progesterone; PKA, protein kinase A; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; LGC, luteal granulosa cell Table II. Gene variants related to insulin resistance in PCOS Gene/References Variant/Locus Subject INS Urbanek et al (72) VNTR PCOS Watherworth et al (77) VNTR PCOS Ferk et al (78) VNTR PCOS INSR Urbanek et al (72) D19S884 PCOS Tucci et al (74) D19S884 PCOS Villuendas et al (79) D19S884 PCOS Siegel et al (80) His1058 PCOS Chen et al (81) His1058 PCOS Jin et al (82) Cys1008 PCOS Mukherjee et al (83) His1058 PCOS Lee et al (84) +176447 PCOS IRS1/2 Urbanek et al (72) IRS PCOS Ehrmann et al (86) Gly972Arg, IRS-1 PCOS Gly1057Asp, IRS-2 PCOS EL Mkadem et al (87) Gly972Arg, IRS-1 PCOS Gly1057Asp, IRS-2 PCOS Villeundas et al (88) Gly972Arg, IRS-1 PCOS Gly1057Asp, IRS-2 Dilek et al (89) Gly972Arg, IRS-1 PCOS Lin et al (90) Gly972Arg, IRS-1, PCOS Ala513Pro, IRS-1 Sir-Petermann et Gly972Arg, IRS-1 PCOS al (91) Baba et al (92) Gly972Arg, IRS-1 PCOS Valdes et al (91) Gly972Arg, IRS-1 PCOS Haap et al (94) Gly972Arg, IRS-1 PCOS Gly1057Asp, IRS-2 PCOS ENPP1 Baba et al (92) K121Q PCOS Heinonen et al (97) K121Q PCOS San Millan et al (98) K121Q PCOS Shi et al (99) K121Q PCOS PPAR[gamma] San Millian et al (98) Pro12Ala PCOS Korhonen et al (100) Pro12Ala PCOS Gu et al (101) Pro12Ala PCOS 1431 C/T PCOS Orio et al (102) Pro12Ala PCOS 1431 C/T PCOS Wang et al (103) Pro12Ala PCOS Hara et al (104) Pro12Ala PCOS Xita et al (105) Pro12Ala PCOS Tok et al (106) Pro12Ala PCOS Yilmaz et al (107) Pro12Ala PCOS Antoine et al (108) Pro12Ala PCOS His447His in exon 6 Control Koika et al (109) Pro12Ala PCOS CAPN 10 Ehrmann et al (110) Genotype 112/121 PCOS Lee et al (111) Haplotype 111, PCOS diplotype 111/121 and 111/111 Haplotype 112, diplotype 112/121 Gonzalez et al (112) UC SNP-44 PCOS Haplotype 1121 Escobar-Morreale et UC SNP-45 PCOS al (113) Haplotype 2111 and 1221 Haddad et al (114) UCSNP -44 PCOS Vollmert et al (115) UC SNP-19 ins/del PCOS UC SNP-56 UC SNP-44 PON1 San Millan et al (98) -108C/T PCOS L55M Q192R ADIPOQ Escobar-Morreale et T45G PCOS al (52) G276T San Millan et al (98) T45G PCOS G276T Xita et al (116) T45G PCOS G276T Zhang et al (117) T45G PCOS G276T Haap et al (94) T45G PCOS Baba et al (118) -11377 PCOS RETN Escobar-Morreale et -420C/G PCOS al (52) Baba et al (118) -420C/G PCOS Urbanek et al (119) -420C/G PCOS Xita et al (120) -179C/G PCOS LEP Oksanen et al (121) Coding region PCOS LEPR Oksanen et al (121) K109R PCOS Q223R K656N 3' UTR Erel et al (122) Q223R PCOS Gene/References Variant/Locus Phenotypic traits INS Urbanek et al (72) VNTR PCOS, hyperandrogenemia Watherworth et al (77) VNTR PCOS Ferk et al (78) VNTR PCOS INSR Urbanek et al (72) D19S884 PCOS Tucci et al (74) D19S884 PCOS Villuendas et al (79) D19S884 PCOS Siegel et al (80) His1058 PCOS in lean Caucasian Chen et al (81) His1058 PCOS Jin et al (82) Cys1008 PCOS Insulin sensitivity Mukherjee et al (83) His1058 PCOS in lean Indians, IR, hyperandrogenemia Lee et al (84) +176447 PCOS IRS1/2 Urbanek et al (72) IRS PCOS Ehrmann et al (86) Gly972Arg, IRS-1 PCOS Insulin and glucose Gly1057Asp, IRS-2 PCOS [down arrow] 2h- glucose EL Mkadem et al (87) Gly972Arg, IRS-1 PCOS [down arrow] Fasting insulin Gly1057Asp, IRS-2 PCOS [up arrow] 2h- glucose Villeundas et al (88) Gly972Arg, IRS-1 PCOS [up arrow] Fasting glucose Gly1057Asp, IRS-2 PCOS [down arrow] 2h- glucose Dilek et al (89) Gly972Arg, IRS-1 PCOS, obese, fasting insulin, IR Lin et al (90) Gly972Arg, IRS-1, PCOS, Metabolic parameters Ala513Pro, IRS-1 Sir-Petermann et Gly972Arg, IRS-1 PCOS al (91) Obesity Baba et al (92) Gly972Arg, IRS-1 PCOS Valdes et al (91) Gly972Arg, IRS-1 PCOS Haap et al (94) Gly972Arg, IRS-1 PCOS Gly1057Asp, IRS-2 PCOS ENPP1 Baba et al (92) K121Q PCOS Heinonen et al (97) K121Q PCOS San Millan et al (98) K121Q PCOS Shi et al (99) K121Q PCOS PPAR[gamma] San Millian et al (98) Pro12Ala PCOS Korhonen et al (100) Pro12Ala PCOS Gu et al (101) Pro12Ala PCOS 1431 C/T PCOS Orio et al (102) Pro12Ala PCOS BMI, glucose, lipid IR 1431 C/T PCOS, [up arrow] BMI, [up arrow] serum leptin Wang et al (103) Pro12Ala PCOS BMI, Reproductive hormones Hara et al (104) Pro12Ala PCOS Insulin sensitivity in Caucasian Xita et al (105) Pro12Ala PCOS Tok et al (106) Pro12Ala PCOS [up arrow] Obesity, [up arrow] Fasting insulin [down arrow] IR Reproductive hormones Yilmaz et al (107) Pro12Ala PCOS [down arrow] Androgens [down arrow] Insulin and IR Antoine et al (108) Pro12Ala PCOS His447His in exon 6 [down arrow] Testosterone [down arrow] Insulin and IR Koika et al (109) Pro12Ala PCOS [down arrow] BMR, hyperandrogenemia CAPN 10 Ehrmann et al (110) Genotype 112/121 PCOS, [up arrow] fasting insulin Lee et al (111) Haplotype 111, PCOS diplotype 111/121 and 111/111 Haplotype 112, [down arrow] PCOS diplotype 112/121 risk Gonzalez et al (112) UC SNP-44 PCOS Haplotype 1121 hypercholesterolemia Escobar-Morreale et UC SNP-45 PCOS al (113) Haplotype 2111 Hirsutism and 1221 Haddad et al (114) UCSNP -44 PCOS Vollmert et al (115) UC SNP-19 ins/del PCOS UC SNP-56 UC SNP-44 PON1 San Millan et al (98) -108C/T PCOS L55M PCOS Q192R PCOS ADIPOQ Escobar-Morreale et T45G PCOS al (52) G276T PCOS San Millan et al (98) T45G PCOS G276T Xita et al (116) T45G PCOS Hyperinsulinemia G276T PCOS [up arrow] Serum adiponectin [down arrow] Insulin Zhang et al (117) T45G PCOS G276T PCOS [down arrow] Insulin, and IR [up arrow] Serum adiponectin Haap et al (94) T45G IR Baba et al (118) -11377 PCOS RETN Escobar-Morreale et -420C/G PCOS al (52) Baba et al (118) -420C/G PCOS Urbanek et al (119) -420C/G PCOS Obesity IR Xita et al (120) -179C/G PCOS BMI LEP Oksanen et al (121) Coding region PCOS LEPR Oksanen et al (121) K109R PCOS Q223R PCOS K656N Low insulin 3' UTR Low insulin Erel et al (122) Q223R PCOS Gene/References Variant/Locus Association INS Urbanek et al (72) VNTR No Watherworth et al (77) VNTR Yes Ferk et al (78) VNTR Yes INSR Urbanek et al (72) D19S884 Yes Tucci et al (74) D19S884 Yes Villuendas et al (79) D19S884 No Siegel et al (80) His1058 Yes Chen et al (81) His1058 Yes Jin et al (82) Cys1008 Yes Yes Mukherjee et al (83) His1058 Yes Lee et al (84) +176447 Yes IRS1/2 Urbanek et al (72) IRS No Ehrmann et al (86) Gly972Arg, IRS-1 No No Gly1057Asp, IRS-2 No Yes EL Mkadem et al (87) Gly972Arg, IRS-1 No Yes Gly1057Asp, IRS-2 No Yes Villeundas et al (88) Gly972Arg, IRS-1 No Yes Gly1057Asp, IRS-2 No Yes Dilek et al (89) Gly972Arg, IRS-1 Yes Lin et al (90) Gly972Arg, IRS-1, No Ala513Pro, IRS-1 Sir-Petermann et Gly972Arg, IRS-1 Yes al (91) Yes Baba et al (92) Gly972Arg, IRS-1 Yes Valdes et al (91) Gly972Arg, IRS-1 No Haap et al (94) Gly972Arg, IRS-1 No Gly1057Asp, IRS-2 No ENPP1 Baba et al (92) K121Q No Heinonen et al (97) K121Q Yes San Millan et al (98) K121Q No Shi et al (99) K121Q No PPAR[gamma] San Millian et al (98) Pro12Ala No Korhonen et al (100) Pro12Ala Yes Gu et al (101) Pro12Ala Yes 1431 C/T Yes Orio et al (102) Pro12Ala No No No 1431 C/T Yes Wang et al (103) Pro12Ala No No Hara et al (104) Pro12Ala No Yes Xita et al (105) Pro12Ala No Tok et al (106) Pro12Ala No Yes Yes No Yilmaz et al (107) Pro12Ala No Yes Yes Antoine et al (108) Pro12Ala No His447His in exon 6 Yes Yes Koika et al (109) Pro12Ala No Yes CAPN 10 Ehrmann et al (110) Genotype 112/121 Yes Lee et al (111) Haplotype 111, Yes diplotype 111/121 and 111/111 Haplotype 112, Yes diplotype 112/121 Gonzalez et al (112) UC SNP-44 Yes Haplotype 1121 Yes Escobar-Morreale et UC SNP-45 Yes al (113) Haplotype 2111 Yes and 1221 Haddad et al (114) UCSNP -44 No Vollmert et al (115) UC SNP-19 ins/del Yes UC SNP-56 Yes UC SNP-44 No PON1 San Millan et al (98) -108C/T Yes L55M No Q192R No ADIPOQ Escobar-Morreale et T45G No al (52) G276T No San Millan et al (98) T45G No G276T No Xita et al (116) T45G No Yes G276T No Yes Yes Zhang et al (117) T45G Yes G276T Yes Yes Yes Haap et al (94) T45G No Baba et al (118) -11377 No RETN Escobar-Morreale et -420C/G al (52) Baba et al (118) -420C/G Yes Urbanek et al (119) -420C/G No No No Xita et al (120) -179C/G No Yes LEP Oksanen et al (121) Coding region No LEPR Oksanen et al (121) K109R No Q223R No K656N Yes 3' UTR Yes Erel et al (122) Q223R No
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