Sex Hormone-Binding Globulin Genetic Variation: Associations with Type 2 Diabetes Mellitus and Polycystic Ovary Syndrome

Abstract

Sex hormone-binding globulin (SHBG) is the primary plasma transport protein for sex steroid hormones and regulates the bioavailability of these hormones to target tissues. The gene encoding SHBG is complex and any of several polymorphisms in SHBG have been associated with alterations in circulating SHBG levels.

Epidemiological studies have revealed that low plasma SHBG levels are an early indicator of insulin resistance and predict the development of type 2 diabetes mellitus (T2DM) in both men and women. Although associations between low SHBG levels and risk of diabetes could be explained by the observation that elevations in insulin suppress hepatic SHBG production, recent studies documenting that the transmission of SHBG-altering polymorphisms are associated with risk of T2DM suggest that SHBG may have a more direct physiologic role in glucose homeostasis. However, the exact mechanism(s) underlying this association is not known.

Non-diabetic women with the polycystic ovary syndrome (PCOS), a common endocrine disorder that is associated with insulin resistance, similarly demonstrate lower levels of SHBG. In light of studies investigating polymorphisms in SHBG and T2DM, our group and others have hypothesized that SHBG may represent a candidate gene for PCOS. In this manuscript, we review studies investigating the association between SHBG polymorphisms and PCOS. In summary, multiple studies in women with PCOS confirm that certain genetic polymorphisms are associated with circulating SHBG levels, but they are not consistently associated with PCOS per se.

INTRODUCTION

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein that transports sex steroid hormones, binding androgens including testosterone and dihydrotestosterone (DHT) with high affinity and estradiol with lower affinity (1;2). SHBG is predominantly synthesized in the liver, and hepatic production is regulated by various hormonal and metabolic factors (3–6). In the past, the predominant physiologic paradigm, the free hormone hypothesis, held that the primary biologic function of SHBG was to regulate the bioavailability of free sex hormones to target tissues (7). However, increasing experimental evidence suggests that SHBG directly activates cell surface signal transduction pathways, modulating the cellular uptake and biologic action of sex steroids (8–11). Thus, alterations in the levels or function of circulating SHBG may contribute to the development of clinical phenotypes through multiple complex physiologic pathways.

Evidence from both in vitro (4;12) and in vivo (5) studies suggests that insulin suppresses hepatic SHBG production. Consequently, low levels of SHBG have been proposed as a biomarker of the metabolic/insulin resistance syndrome in overweight men and women and may predict the development of T2DM (13–15). Although metabolic factors directly influence SHBG levels, circulating levels of the glycoprotein are also partially determined by genetic variation (16–18). Importantly, two recent studies have demonstrated that single nucleotide polymorphisms(SNPs) in the SHBG gene are associated with levels of SHBG and predict future risk of T2DM (19;20). Such findings strengthen the hypothesis that alterations in SHBG may contribute to the etiology of T2DM, and potentially other disorders characterized by insulin resistance.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder among women of reproductive age, affecting 5–10% of this population (21;22), and is characterized by chronic anovulation and hyperandrogenism (23). Moreover, women with PCOS demonstrate an increased burden of insulin resistance that is thought to be intrinsic to the syndrome (24) and are at increased risk of T2DM (25). PCOS women also have lower serum levels of SHBG compared with normal women (26), and suppressed SHBG levels worsen the biochemical hyperandrogenemia characteristic of the syndrome by increasing levels of bioavailable androgens. Previous family-based studies support a significant genetic predisposition to PCOS (27;28); although the specific genes contributing to the syndrome remain to be definitively elucidated. In light of evidence suggesting that alterations in SHBG may contribute to the metabolic disturbances central to the pathophysiology of PCOS, SHBG represents an attractive candidate gene for the disorder.

Biochemical Structure of SHBG Glycoprotein

SHBG is a glycoprotein comprised of two identical, noncovalently-bound subunits (2). In humans, each SHBG monomer subunit consists of a 373 amino acid polypeptide with three oligosaccharide side chains and two disulfide bonds (2). Each SHBG subunit contains a steroid binding site capable of binding DHT, testosterone, or estradiol, such that the mature SHBG homodimer has two distinct steroid binding sites (29). Moreover, each monomer contains two β-sheets which are essential in the dimerization of the mature SHBG glycoprotein. Specifically, eight hydrogen bonds are formed across the interface of the β-sheets such that two continuous 14-stranded β-sheets are formed in the mature homodimer (29;30).

Similar to other steroid hormone-binding glycoproteins such as cortisol-binding globulin or thyroxine-binding globulin, mature SHBG contains oligosaccharide side chains, and the structural organization of the carbohydrate moieties are specific to each binding glycoprotein (31). Specifically, each subunit of the SHBG homodimer is characterized by three oligosaccharide moieties, an O-linked glycosylation site at Thr7, and N-linked sites at Asn 351 and Asn 367 (32–34). Although the oligosaccharide side-chains on SHBG do not appear to be critical for the glycoprotein’s steroid-binding activity (34), similar to the biologic function observed in other glycoproteins, SHBG glycosylation may be important in the glycoprotein’s interaction with specific cell-surface receptors (35).

In addition to binding sex steroids, the SHBG homodimer itself serves as a ligand for a specific, high affinity receptor (R_SHBG) present on the plasma membranes of target cells (8;10;11;36;37). Only steroid-free SHBG appears to bind to R_SHBG; however, once SHBG is bound to the receptor, sex steroids can then activate the anchored SHBG-R_SHBG complex (8). Moreover, adding additional complexity to the system, not all steroids that bind to the SHBG-R_SHBG complex function as agonists; some are antagonists (8). Moreover, some steroids such as DHT may function as either an agonist or antagonist for the system, depending on the specific target cell type (8;38). Although the full downstream effects of SHBG-R_SHBG complex activation remain unclear, complex activation appears to affect target cell growth in addition to modulating the transcriptional activity of classic intracellular steroid hormone receptors (8).

SHBG may also actively participate in the uptake of sex steroids by target tissues through interactions with megalin, an endocytic receptor distinct from R_SHBG (9). Although the uptake of SHBG-bound sex hormones via the megalin-mediated pathway is controversial (39), such findings support an expanded role of SHBG in sex steroids physiology.

SHBG Gene Structure and Splice Variants

The SHBG gene, located on the chromosome 17p12→p13, consists of eight exons separated by seven small introns (2;40;41). Exon 1 encodes for the nacent protein’s 29 amino acid secretion signal polypeptide (2), while the remaining exons [2–8] encode two contiguous laminin G–like (LG) domains (41). The amino-terminal LG domain encoded by exons 2–4 contains the steroid-binding site, the dimer interface, and several cation-binding sites (42). A ten amino acid sequence (residues 48–57) within exon 3 appears to correspond to the R_SHBG-binding domain (43).

Although hepatocytes are the primary source of plasma SHBG (44), extrahepatic tissues, including testis, prostate, ovary, endometrium, breast, placenta and hypothalamus also express SHBG mRNA in humans (45–51). In fact, recent evidence suggests that the transcriptional control of SHBG gene expression is extremely complex and is regulated by at least three distinct promoters (P_L, P_T, and P_N) which are expressed differentially in various human tissues (52).

Activation of the downstream promoter (P_L), results in the production of the most common SHBG mRNA transcript [exon1L-8] (52). The exon IL-8 transcript is predominantly expressed in hepatocytes and encodes for all eight exons present in the SHBG gene. P_L activation in the testis results in an identical eight-exon mRNA; however, distinct post-translational processing of the testicular transcript results in the production of androgen binding protein (ABP) instead of mature SHBG (45;53). In addition to the liver and testis, the 1L-8 mRNA transcript is also expressed in the human prostate, breast and regions of the brain (52). In the testis, activation of a second SHBG promoter (P_T), located 1.9 kb upstream of P_L, produces a second major mRNA transcript (45;52). In addition to possessing an unique 5′ end amino acid sequence (exon 1T), the second testicular transcript also lacks exon 7(45;52). Recently, Nakhla and colleagues described a third SHBG gene promoter (P_N), located within intron 1 of the adjacent FXR2 gene (52). Similar to P_L and P_T transcripts, P_N transcripts possess a distinct first exon (1N). Differential activation of the three promoters triggers alternative splicing of SHBG exons which, in turn, may result in the expression of at least 19 unique SHBG transcripts (52). Furthermore, the pattern of SHBG transcript expression differs in normal tissues with P_L-, P_T-, and P_N- derived transcripts being most abundantly expressed in the liver, testis, and prostate, respectively (52). Interestingly, alternative splicing of SHBG is more pronounced in certain cancer cell lines compared with normal tissues (52) (Figure 1). Although Nakhla and colleagues hypothesize that only certain P_L-derived transcripts produce stable SHBG isoforms, the potential biologic significance of alternatively spliced SHBG gene transcripts remains unclear (52).

Figure 1. Structure, size, and tissue distribution of the major (IL-8) and alternatively spliced SHBGtranscripts arising from the downstream promoter, P_L.

The P_L promoter directs the expression of amajor mRNA transcipt encoding for SHBG/APB in addition to multiple other independent transcripts which result from alternative splicing of exons 4,5,6 and/or 7. Rectangles represent exons (E). Conserved exons are labeled in black; alternatively spliced exons are labeled in blue. Alternatively spliced transcripts are identified by missing exons. SHBG has three glycosylation sites (Thr7 [O-linked]; Asn 351, Asn 367 [N-linked]) which are identified by ▼. The differential expression of each transcipt in normal human tissues and specific human cancer cell lines (HepG2 –hepatocellular carcinoma; LNCaP – prostate adenocarcinoma; MCF-7 - breast cancer) are identified on the right. ABP – androgen binding protein; SHBG – sex hormone-binding globulin. Adapted from Nakhla et al. BMC Molecular Biology. 2009;10:37.

Regulation of SHBG Expression by Insulin and Monosaccharides

Hepatic SHBG biosynthesis is regulated by multiple metabolic and hormonal factors including sex steroids, thyroxine, prolactin, and insulin (4;12;54;55). Supporting a potential biologic mechanism for the observed associations between hyperinsulinemia, hyperglycemia and alterations in SHBG (56), in vitro experiments suggest that insulin suppresses SHBG production in the human hepatoma (HepG2) cell line (4;12). However, this observation is controversial as other investigators have suggested that the suppressive effects of insulin are not specific to SHBG and likely represent a more global reduction in hepatic protein secretion under specific culture conditions (57). Proposing an alternative hypothesis, Selva and colleagues demonstrated that hepatic SHBG transcription is primarily responsive to levels of monosaccharides rather than to insulin (6). Specifically, exposure to glucose or fructose induced hepatic lipogenesis and led to reduced SHBG production by HepG2 cells via down-regulation of hepatocyte nuclear factor-4α (HNF-4α), a transcription factor that plays a critical role in controlling the SHBG promoter (6). Although insulin did not appear to influence SHBG production in the series of well-designed in vitro experiments conducted by Selva et al., such findings must also be considered in light of in vivo human experiments demonstrating that the acute suppression of pancreatic insulin secretion by diazoxide results in a significant increase in circulating levels of SHBG (5). Although the controversies regarding the role of insulin in hepatic SHBG production warrant further investigation, collectively these studies support potential mechanisms through which plasma SHBG levels are linked to metabolic disturbances resulting from excess carbohydrate consumption (58).

Associations of Sex Hormones, SHBG and Type 2 Diabetes Mellitus

Similarly, previous observational studies support associations between levels of endogenous sex hormones and/or SHBG and the development of T2DM. In a large systemic review and meta-analysis, Ding and colleagues describe a consistent sexual dimorphism in the relationship between testosterone and diabetes risk. Specifically, lower levels of testosterone are associated with diabetes in men whereas higher testosterone levels are associated with the disorder in women (59). Results of animal studies not only support the sexual dimorphism observed in humans but also point to sex-specific causal mechanisms between testosterone and the development of diabetes (60;61). On the other hand, the relationship between estradiol and T2DM is less certain. Although Ding et al. observed an association with elevated estradiol levels and T2DM in both men and women independent of body mass index (BMI) (59), results of animal studies suggest that estradiol increases pancreatic islet cell survival, potentially protecting against diabetes (62;63).

Consistent with both the long held free hormone hypothesis and newer evidence suggesting that SBHG directly modulates the signal transduction and, consequently, biologic action of sex steroids (8–10), alterations in circulating levels of SHBG may mediate the relationships between sex steroids and T2DM. Indeed, low circulating levels of SHBG appear to be a strong predictor of the risk of T2DM in both women and men (19;20;59;64;65) and may herald the onset of abnormalities in glucose homeostasis (14;15;20;66). Moreover, associations between levels of SHBG and incident T2DM in both men and women appear to be independent of levels of endogenous sex hormones (64;65).

Although such observations may be explained by reverse causation, with subtle elevations in insulin or monosaccharides influencing SHBG levels long before the development of clinical disease, two recent studies demonstrating that SHBG SNP genotypes are directly associated with both levels of circulating SHBG and the risk of T2DM suggest a causal role of SHBG in the disorder (19;20). Using a nested case-control study design involving post-menopausal women from the Women’s Health Study and men enrolled in the Physicians’ Health Study, Ding and colleagues prospectively demonstrated that decreased levels of SHBG are associated with increased risk of T2DM (20). Furthermore, carriage of a variant allele for either of two SHBG SNPs, rs6257 and rs6257, influenced diabetes risk in a manner consistent with their effects on plasma SHBG. Specifically, compared with homozygotes for the respective wild-type allele, carriage of a variant allele in rs6257 was associated with lower levels of SHBG and higher risk of type 2 T2DM. Conversely, carriage of variant rs6259 was associated with higher levels of SHBG and lower risk of T2DM (20). In a subsequent meta-analysis involving 27,657 patients with T2DM and 58,481 controls, Perry and colleagues observed a significant association between the presence of T2DM and the genotype for a third SNP in SHBG, rs1799941 (19). Specifically, the SHBG raising allele (A) was associated with a reduced risk of T2DM, and the association between rs1799941 genotype remained significant after controlling for age, sex, and BMI (19). These two studies strongly support a physiologic role of SHBG in the etiology of T2DM; however, specific biologic mechanisms remain to be definitively elucidated.

SHBG Variants and Association with PCOS

Despite uncertainty regarding specific metabolic pathways through which SHBG influences insulin sensitivity and the risk of T2DM, SHBG has been proposed as an attractive candidate gene for PCOS (67;68). Specifically, our group and others have hypothesized that genetic variation in SHBG levels may contribute to at least two of metabolic derangements that are central to the disorder, namely insulin resistance and hyperandrogenemia. Consequently, several specific variants of the SHBG gene have been studied for association with PCOS, including a pentanucleotide (TAAAA)_n repeat polymorphism, rs1799941 (−67G>A in the 5′ untranslated region), rs6257 (IVS1-17T>C); rs6259 (Ex8 +6G>A(D356N)) and rs727428 (1121bp 3′of STP T>C) (Figure 2).

Figure 2. Location of polymorphisms of interest within the SHBG gene (17p12–13).

Rectangles represent exons (E). Black diamonds represent coding SNPs; gray diamonds represent 5′-UTR SNP; unfilled diamonds represents intronic SNPs; triangles represents promoter SNP. Numbers refer to relativepositions along chromosome 17. P_{L -} SHBG gene downstream promoter; P_T - SHBG gene upstream promoter. (TAAAA)_n is associated with SHBG levels and PCOS. rs1799941 is associated with SHBG levels, androgen levels and risk of T2DM. rs6257 is associated with SHBG levels and risk of T2DM.P156L is associated with extremely low SHBG levels and severe hyperandrogenism. rs6259 is associated with SHBG levels and risk of T2DM. rs727428 is associated with SHBG levels. PCOS – polycystic ovary syndrome; SNP – single nucleotide polymorphism; SHBG – sex hormone-binding globulin; T2DM – Type 2 diabetes mellitus. Adapted from Xita N, Tsatsoulis A. Molecular and Cellular Endocrinology. 2010;316:60–65.

Polymorphisms in a pentanucleotide (TAAAA)_n repeat located in the 5′ promoter region of SHBG have been associated with transcriptional activity of the gene in vitro (69). In the general population, the SHBG gene contains at least 6–10 TAAAA repeat, and SHBG transcriptional activity is lower in HepG2 cell lines transfected with SHBG promoter transcripts containing six TAAAA repeats compared with cells containing 7–10 repeats (69). Although at least six studies have investigated the association of this polymorphism with PCOS or clinical hyperandrogenism, the results have been inconclusive (26;67;68;70–72).

In a case-control study conducted by Liu et al. involving 187 Chinese women with PCOS and 176 controls, the TAAAA_n polymorphism was neither a determinant of PCOS nor a predictor of serum SHBG levels (72). Ferk and colleagues compared the frequency of TAAAA_n alleles in a cohort of Slovenian women according to PCOS status (26). Although this group, again, did not find that TAAAA_n repeat frequency was significantly different in PCOS women (n=123) compared with normal controls (n=110) (26), the investigators did report that serum SHBG values were strongly influenced by the polymorphism in both groups. In a cohort of 303 French hirsute women, Cousin et al. found that SHBG levels were significantly higher in women homozygous for six-TAAAA repeat sequence compared with those women homozygous for the nine-repeat polymorphism; however, the analysis was somewhat limited by the large number of TAAAA genotypes present in their population (68). Despite the lack of association in other ethnic/racial populations, the (TAAAA)_n polymorphism was significantly associated with PCOS susceptibility in a study of Greek women (185 with PCOS and 324 normal controls) conducted by Xita et al (67). Moreover, in the Greek study, PCOS women with longer (TAAAA)_n genotypes had lower SHBG levels than PCOS women with shorter repeats (67). Using the same cohort of Greek women, Xita and colleagues subsequently demonstrated that polymorphisms in two other genes, cytochrome P450 [(CYP19)(TTTA)_n] and the androgen receptor [AR(CAG)_n] synergistically influenced the hyperandrogenic phenotype of PCOS with SHBG (TAAAA)_n genotype variants (70;71). Such findings support the hypothesis that variants in multiple genes likely contribute to the clinical development of PCOS.

Multiple SHBG SNPs have been associated with circulating levels of SHBG in women (73–75), and thus represent potential candidate loci for PCOS. The first SNP, rs1799941, is located in the 5′ UTR region of the SHBG gene, 8 nucleotides upstream of the transcription start site (41). In the study by Riancho et al., SHBG levels were significant higher in women with A/A genotypes for rs1799941 compared with individuals with G/G genotypes (75). This same SNP (rs1799941) was subsequently shown by Perry and colleagues to not only influence circulating SHBG levels but convey risk for T2DM in the large study involving both men and women (19).

Riancho et al. also investigated the relationship between genotypes for two additional SHBG SNPs, rs6257 and rs6259, and SHBG levels (75). The rs6257 SHBG polymorphism (D365N) is located 17 bp upstream of exon 2, while rs6259 is located in exon 8 and encodes for a non-synonymous amino acid change (D327N) (76;77). The latter SNP introduces an additional site for N-glycosylation in the mature SHBG protein (76). Although the addition of a carbohydrate moiety as a result of the rs6259 polymorphism does not affect the binding of steroids to SHBG (76), it may reduce the plasma clearance of SHBG, leading to a modest accumulation of the variant protein in the blood (78). As a result, the rs6259 substitution may be associated with higher SHBG levels in variant allele carriers (68), potentially protecting women from the development of hyperandrogenism. However, despite reported associations of D327N with increased SHBG levels in multiple studies involving women (20;74) and the association of rs6259 genotype and the risk of 2TDM in the large study by Ding and colleagues (20), the rs6259 polymorphism did not appear to influence susceptibility to PCOS according to a study conducted in Czech women including 248 PCOS patients and 109 healthy control women (79).

An additional SHBG SNP of interest, rs727428, lies 1.1 kb beyond the 3′-end of the gene, with the minor allele for rs727428 being associated with lower SHBG levels. This region is highly conserved across species, suggesting a potentially functional role for rs727428 in the regulation of SHBG gene transcription (73). We recently investigated the associations of rs727428 and three other SHBG SNPs (rs1799941, rs6257, and rs6259) with PCOS in a family-based linkage study involving 430 families with female offspring affected by PCOS (unpublished data). Although we found that serum SHBG levels were significantly associated with minor allele frequency for both rs727429 and rs727428 even after controlling for the influence of other potential confounding variables, including body mass index (BMI), unbound testosterone, and estimates of insulin resistance, we did not observe statistically significant associations between SNP genotype and serum SHBG levels for rs6257 or rs6259 in our analyses. We also did not find any evidence of linkage or association between any of the four SNPs of interest and PCOS using a family-based method.

Although less common SHBG variants are unlikely to contribute to the phenotype for most women with PCOS, understanding the biologic mechanisms linking rarer SHBG variants and clinical phenotype may shed additional light on the pathophysiology of the disorder. For example, the rare missense variant in exon 4, P156L, allows for normal steroid ligand binding; however, P156L results in abnormal SHBG glycosylation and, consequently, inefficient secretion of SHBG (41;42). Hogeveen and colleagues first described the P156L mutation in a 27-year French women presenting with severe hyperandrogenism and very low serum SHBG who was found to be homozygous for the variant allele (42). The same group subsequently described the heterozygous state in four women among a cohort of 294 women with hirsutism or ovarian dysfunction; the P156L variant allele was not identified among the 88 normal women and 53 women with 21-hydroxylase deficiency who were also screened (42).

Conclusions

The transcriptional control of the SHBG gene is complex, and several polymorphisms in the gene appear to influence circulating levels of SHBG. Individuals with both T2DM and women with PCOS, two disorders characterized by insulin resistance, demonstrate lower circulating levels of SHBG compared with normal control subjects. Although the association between low levels of SHBG and insulin resistance could be explained in part by the inhibitory effects of insulin on hepatic SHBG production, several reports supporting associations between genetic variants in SHBG that affect circulating levels of the protein and future risk of T2DM may point to a more direct role of SHBG in glucose homeostasis. In light of the central role of insulin resistance in both T2DM and PCOS, SHBG is an attractive candidate gene for PCOS. However, several studies have now failed to show strong consistent evidence for association between any of the common variants in the SHBG gene and the development of PCOS. The observed discrepancies between studies examining the associations between SHBG SNP transmission and risk of T2DM and PCOS may be explained by the significant genetic heterogeneity responsible for both disorders.