Interactions between human plasma sex hormone-binding globulin and xenobiotic ligands

Human sex hormone-binding globulin (SHBG) binds sex steroids with high affinity. In plasma, the number of SHBG steroid-binding sites far exceeds the molar concentrations of sex steroids, and will accommodate other ligands such as phytoestrogens and fatty acids. We have therefore developed a screening assay to identify ligands for SHBG, which exist in our diet or environment. This assay allows the binding of potential ligands to SHBG to be assessed under physiological conditions, and is sensitive to the effects of plasma constituents. Several classes of endocrine active compounds were tested, including hydroxy-polychlorinated biphenyls (HO-PCBs), phthalate esters, monoesters, chlorinated pesticides, as well as synthetic estrogens and phytoestrogens. The relative binding affinities (RBAs) of various compounds to SHBG were determined in competitive displacement assays, by comparison with 17 beta-estradiol (RBA=100). Synthetic estrogens bound SHBG with RBAs of 0.4 (ethinylestradiol)-0.2 (diethylstilbestrol), while some phytoestrogens bound with RBAs of 0.12 (coumestrol)-0.04 (naringenin). Many compounds did not bind to SHBG with sufficient affinity to allow RBA measurements, and these include: several phytoestrogens, such as genistein and kaempferol, polychlorinated biphenyls, phthalate esters and monoesters. Of nine HO-PCB congeners tested only 4-OH-2', 3', 4', 5'-tetraCB and 4-OH-2, 2', 3', 4', 5'-pentaCB bound SHBG in undiluted serum with RBAs of 0.05 and 0.11. Although all test compounds bound to SHBG with much lower affinity than endogenous sex steroids, these interactions may be physiologically relevant in situations where plasma SHBG levels are high and endogenous sex steroid levels are low, such as in pre-pubertal children and women taking oral contraceptives.     

Plasma variation of corticosteroid-binding globulin and sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) circulate in plasma and bind their cognate ligands with high affinity, offering a steroid delivery system to target tissues by a variety of mechanisms. Analysis of these steroid-binding proteins is gaining importance in the clinical setting, although more information is warranted on their diurnal and biological variation. This study shows that plasma SHBG (in normal subjects) exhibits little diurnal or biological variation over the 30 day period studied, in contrast to CBG, where plasma levels peak in the early afternoon. This leads to attenuation of the diurnal free cortisol level rhythm compared to total cortisol. We also show that plasma CBG is significantly lower in male subjects with the metabolic syndrome compared to age-matched lean counterparts, and may therefore act as a surrogate marker of insulin resistance. The consequence of lower levels of CBG in these obese male subjects is reflected by higher levels of circulating free cortisol, potentially offering a more favourable environment for adipogenesis.     

Sex hormone-binding globulin and thyroxine-binding globulin levels in premature thelarche

Premature thelarche is defined as the isolated development of breast tissue in girls less than 8 years of age. Although breast development is an estrogen-dependent process, these girls do not have elevated serum estrogen levels, and the hormonal basis for their condition is unclear. We studied the levels of two estrogen-dependent transport proteins, sex hormone-binding globulin (SHBG) and thyroxine-binding globulin (TBG), in order to determine if there was evidence for a more subtle estrogen effect in girls with premature thelarche. SHBG levels in girls with premature thelarche were not significantly different from those of prepubertal girls of the same ages and were significantly lower than those in girls undergoing pubertal development at the appropriate age (P less than 0.05) and in normal women (P less than 0.001). There was no statistically significant difference in TBG levels between the girls with premature thelarche and prepubertal controls. There was also no significant difference in TBG levels between prepubertal girls and girls in early puberty. In contrast, women had TBG levels that were significantly lower than those in all girls studied. We conclude that the estrogen exposure (whether endogenous or exogenous) of girls with premature thelarche is less than that of girls in early true puberty and similar to that of other prepubertal girls. Further, changes in serum TBG are not as sensitive an indicator of estrogen effect as is breast development or changes in SHBG. This study also suggests that large amounts of exogenous estrogens are not an element in the development of premature thelarche.     

Daily variations of plasma sex hormone-binding globulin binding capacity, testosterone and luteinizing hormone concentrations in healthy rested adult males

In this study the daily variations of plasma sex hormone-binding globulin (SHBG) binding capacity were measured together with plasma testosterone and luteinizing hormone (LH) concentrations in 7 healthy rested adult males. Plasma SHBG-binding capacity demonstrated a significant circadian rhythm (acrophase = 2.06 p.m.; mesor = 0.35 +/- 0.6 ng testosterone bound/100 ml; amplitude = 17% of the mesor). Plasma testosterone also showed a circadian rhythm (acrophase = 7.02 a.m.; mesor = 4.38 +/- 0.67 ng/ml; amplitude = 18% of the mesor). The free testosterone index (or the ratio between plasma testosterone and SHBG-binding capacity) was not correlated with plasma LH levels. In our hands this last parameter did not vary according to a circadian pattern. These data are discussed in terms of a feedback mechanism controlling the pituitary-testis axis regulation.     

Interrelations between sex hormone-binding globulin (SHBG), plasma lipoproteins and cardiovascular risk

The incidence of coronary artery disease is significantly higher in men than in women, at least until menopause. This gender difference could be explained by the action of sex steroids on the lipoprotein profile. In prepubertal children, high-density lipoprotein (HDL) cholesterol and triglyceride levels are similar between sexes, while adult men have generally lower HDL cholesterol and higher triglyceride levels than premenopausal adult women. Most cross-sectional studies have reported that sex hormone binding globulin (SHBG) and testosterone levels correlate positively with HDL cholesterol levels between sexes. Thus SHBG by modulating the balance in the biodisposal of testosterone and estradiol, might have a profound effect on the risk of cardiovascular disease. However, adjustment for body weight and body fat distribution weakens the association between SHBG, testosterone and HDL cholesterol. The negative correlation of fasting insulin with SHBG and HDL cholesterol levels in both sexes, and some evidence that insulin is an inhibitor of SHBG production in vitro, has suggested that hyperinsulinism might negatively regulate SHBG and HDL levels. It remains to be determined whether the inverse relationship between SHBG and insulin levels is coincidental or has a causal effect on the increase of atherosclerosis. Decreased SHBG has been shown to be predictive of the incidence of non-insulin-dependent diabetes mellitus in women but not in men, and of subsequent development of cardiovascular disease and overall mortality in postmenopausal women. SHBG is an index of androgenism in women and of insulin-resistance in both sexes, and might be useful in epidemiological studies of cardiovascular risk. However, in men, SHBG is not predictive of the occurrence of cardiovascular disease. Whether SHBG might have an intrinsic protective effect on the arterial wall through SHBG-receptors is still highly speculative.     

Diverse roles for sex hormone-binding globulin in reproduction

Sex hormone-binding globulin (SHBG) transports androgens and estrogens in blood and regulates their access to target tissues. Hepatic production of SHBG fluctuates throughout the life cycle and is influenced primarily by metabolic and hormonal factors. Genetic differences also contribute to interindividual variations in plasma SHBG levels. In addition to controlling the plasma distribution, metabolic clearance, and bioavailability of sex steroids, SHBG accumulates in the extravascular compartments of some tissues and in the cytoplasm of specific epithelial cells, where it exerts novel effects on androgen and estrogen action. In mammals, the gene-encoding SHBG is expressed primarily in the liver but also at low levels in other tissues, including the testis. In subprimate species, Shbg expression in Sertoli cells is under the control of follicle-stimulating hormone and produces the androgen-binding protein that influences androgen actions in the seminiferous tubules and epididymis. In humans, the SHBG gene is not expressed in Sertoli cells, but its expression in germ cells produces an SHBG isoform that accumulates in the acrosome. In fish, Shbg is produced by the liver but has a unique function in the gill as a portal for natural steroids and xenobiotics, including synthetic steroids. However, salmon have retained a second, poorly conserved Shbg gene that is expressed only in ovary, muscle, and gill and that likely exerts specialized functions in these tissues. The present review compares the production and functions of SHBG in different species and its diverse effects on reproduction.     

Influence of glycosylation on the clearance of recombinant human sex hormone-binding globulin from rabbit blood

Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein that binds sex steroids with high affinity. Variations in hSHBG glycosylation contribute to its electrophoretic microheterogeneity, but the functional significance of different SHBG glycoforms is unknown. Carbohydrates may influence the biological activities and half-lives of glycoproteins and we have examined how oligosaccharides at specific sites influence the plasma clearance of hSHBG in vivo. To accomplish this, fully-glycosylated hSHBG, or hSHBG mutants lacking specific oligosaccharides chains, were expressed in Chinese hamster ovary (CHO) cells and purified by immunoaffinity chromatography. The purified recombinant proteins were then biotinylated to study their plasma half-lives after intravenous injection into rabbits. When compared to hSHBG isolated from serum, recombinant hSHBG migrates with a slightly larger average molecular size during denaturing polyacrylamide gel electrophoresis. This is due to a greater proportion (33–39% vs. 3%) of more highly branched N-linked oligosaccharides on the recombinant proteins. When injected into rabbits, the disappearance of recombinant hSHBG showed two exponential components, as previously shown for natural hSHBG in the same animal model. The mean±S.E.M. plasma half-lives of recombinant hSHBG (t_1/2 0.11±0.03 h and t_1/2β 18.94±1.65 h) are shorter than previously measured for natural hSHBG (t_1/2 3.43±0.72 h and t_1/2β 38.18±7.22 h) and this is likely due to differences in the composition of their N-linked oligosaccharides. An O-linked chain at Thr⁷ does not influence the plasma clearance of hSHBG in the presence or absence of N-linked carbohydrates at Asn³⁵¹ and Asn³⁶⁷. However, a 1.5–1.6 fold (p<0.03) increase in plasma half-life of variants lacking both N-glycosylation sites was observed and this is probably due to the fact these variants are not recognized by the asialoglycoprotein receptor-mediated clearance system. Removal of either N-glycosylation consensus site also increased (p<0.0001) the plasma half-life of hSHBG by 2.3–2.4 fold. Thus, the metabolic clearance of hSHBG appears to be determined by the number of N-linked oligosaccharides rather than their location.     

Tissue uptake of human sex hormone-binding globulin and its influence on ligand kinetics in the adult female rat.

The distribution of human sex hormone-binding globulin (hSHBG) and its influence upon the kinetics of its ligands were assessed in the adult female rat, which lacks a comparable protein in serum. Purified hSHBG was administered i.v. to adult female rats as a single bolus. The plasma disappearance rate of immunoreactive hSHBG had one component with a half-life of 15 h. The estradiol (E2) binding activity of serum attributable to hSHBG was elevated 2-fold; during a continuous infusion of E2, hSHBG increased E2 serum levels above those of control infused animals. Treatment with hSHBG did not modify the plasma clearance of endogenous E2, but accelerated the disappearance rate of 5 alpha-dihydrotestosterone (DHT). In animals injected with a tracer dose of radioactive steroids, pretreatment with hSHBG increased uterine and oviductal accumulation of E2- but not DHT-associated radioactivity. This effect was specific to some E2 target tissues since hSHBG did not alter the concentration of E2- or DHT-associated radioactivity in the hypophysis, liver, diaphragm, or brain. Treatment with anti-E2 antibodies, which elevated E2 binding activity in serum, decreased the accumulation of E2-associated radioactivity in uterus and oviduct. Immunofluorescent localization of hSHBG revealed intense labeling of the uterine and oviductal epithelium. We conclude that this foreign hormone-binding globulin introduced in serum at concentrations that have minimal circulating reservoir effect on E2 can reach intracellular domains and affect the concentration of this ligand in target tissues.     

Influence of changes in the concentration of sex hormone-binding globulin in human serum on the protein binding of the contraceptive steroids levonorgestrel, 3-keto-desogestrel and gestodene

The serum protein binding of levonorgestrel, gestodene and 3-keto-desogestrel has been determined during several clinical studies with different oral contraceptive formulations and one in vitro study. The results of these studies were combined in order to assess the relation between changes in the concentration of sex hormone-binding globulin (SHBG) and the effect on the free fraction of the progestins as well as on their distribution with respect to the binding proteins albumin and SHBG. Although marked differences in protein binding were seen for the three progestins at low concentrations of SHBG, these differences became less pronounced at higg levels of SHBG which were reached during established oral contraceptive therapy. A nonlinear relation could be shown for either the free or the protein-bound fraction of the progestins and the concentration of SHBG in the serum, respectively.     

Changes in serum sex hormone-binding globulin and in serum non-sex hormone-binding globulin-bound testosterone during prepuberty in boys

Much evidence suggests that sex hormone-binding globulin (SHBG) influences the delivery of sex steroids to cells, probably by playing an important role in the distribution of serum sex hormones between SHBG-bound, albumin (HSA)-bound and free fractions. Recent evidence also suggests that HSA-bound testosterone (T), the major constituent of non-sex hormone-binding globulin-bound T, is biologically important. To examine the potential exposure of peripheral tissues to T during prepubertal years, the serum concentration of SHBG as well as the distribution of serum T in SHBG-bound, HSA-bound, free and non-SHBG-bound fractions was studied in 80 normal boys aged 0.5-14 yr, all at Tanner's stage G1 of sexual development. A gradual decrease in serum SHBG as a function of age was found without significant changes in the Ka of SHBG-dihydrotestosterone association. While regression analysis of serum total T vs age showed a 2.6-fold increase from 0.5 to 14 yr of age, those of non-SHBG-found, HSA-bound and free T vs age showed 8- to 9-fold increases during the same period. On the other hand, SHBG-bound T had only a 1.9-fold increase. Expressed as a function of serum total T, non-SHBG-bound T increased from 6.6 to 30.4%, the relative increment being greater for HSA-bound T than for free T. It is concluded that, with advancing age, there is a progressive increase in the T exposure of all tissues in normal prepubertal boys. It is speculated that, at the level of the central nervous system, this increase in serum bioavailable T could induce maturative changes in brain cells that result in the onset of puberty in normal boys.     

Characterization of obese women with reduced sex hormone-binding globulin concentrations

Factors influencing sex-hormone binding globulin (SHBG) concentrations in obesity are poorly understood. Preliminary observations suggest that dietary lipids may be involved and there are data confirming a direct inhibiting effect of insulin. Since only some obese subjects show lowered SHBG levels, we performed this study with the aim of defining obese women with low SHBG (LSO) (2 SD above normal values) in comparison with those presenting normal globulin concentrations (NSO). These groups were selected from a larger group of obese women with a history of normal menses and aged less than 40 years. An age-matched group of normal weight healthy women served as controls. Both LSO and NSO had similar body mass index and percentage body fat, but the waist to hip girth ratio (WHR), an index of body fat distribution, was significantly higher in LSO (0.88 +/- 0.04) than in NSO (0.81 +/- 0.09; P less than 0.05). Gonadotropin and androgen concentrations were similar in both groups, whereas estrone (E1) levels were higher in LSO (32.8 +/- 15.8 pg/ml) than in NSO (19.4 +/- 6.2 pg/ml; P less than 0.05; controls: 23.5 +/- 7.8 pg/ml; P less than 0.05). Moreover, compared to NSO, LSO women had significantly higher glucose-stimulated insulin and C-peptide levels. Partial regression analysis revealed significant correlation coefficients between SHBG, stimulated insulin values (r = -0.38; P less than 0.05) and WHR (r = 0.40; P less than 0.005). Therefore, compared to NSO, LSO women have distinctive clinical and endocrine characteristics, namely more pronounced hyperinsulinemia, higher E1 concentrations and a central type body fat distribution.     

Associations of sex hormone-binding globulin and testosterone with genome-wide DNA methylation

Background

Levels of sex hormone-binding globulin (SHBG) and the androgen testosterone have been associated with risk of diseases throughout the lifecourse. Although both SHBG and testosterone have been shown to be highly heritable, only a fraction of that heritability has been explained by genetic studies. Epigenetic modifications such as DNA methylation may explain some of the missing heritability and could potentially inform biological knowledge of endocrine disease mechanisms involved in development of later life disease. Using data from the Avon Longitudinal Study of Parents and Children (ALSPAC), we explored cross-sectional associations of SHBG, total testosterone and bioavailable testosterone in childhood (males only) and adolescence (both males and females) with genome-wide DNA methylation. We also report associations of a SHBG polymorphism (rs12150660) with DNA methylation, which leads to differential levels of SHBG in carriers, as a genetic proxy of circulating SHBG levels.

Results

We identified several novel sites and genomic regions where levels of SHBG, total testosterone, and bioavailable testosterone were associated with DNA methylation, including one region associated with total testosterone in males (annotated to the KLHL31 gene) in both childhood and adolescence and a second region associated with bioavailable testosterone (annotated to the CMYA5 gene) at both time-points. We also identified one region where both SHBG and bioavailable testosterone in males in childhood (annotated to the ZNF718 gene) was associated with DNA methylation.

Conclusion

Our findings have important implications in the understanding of the biological processes of SHBG and testosterone, with the potential for future work to determine the molecular mechanisms that could underpin these associations.

     

Dehydroepiandrosterone sulfate and other possible influencing factors that modulate sex hormone-binding globulin levels in the hirsute patient.

This study was designed to investigate the most important factors affecting serum concentrations of sex hormone-binding globulin (SHBG) in women with hirsutism. We compared endocrine profiles based on biochemical measurements of LH, FSH, oestradiol, testosterone (T), prolactin, 17-hydroxy-progesterone, dehydroepiandrosterone sulphate (DHEAS), SHBG, cortisol and insulin in the follicular phase in 32 healthy women and 52 patients. The study group was subdivided according to SHBG levels into Group A (low level) and Group B (high level). Significant differences between Groups A and B were found in DHEAS and T levels, but not in body mass index or insulinaemia. There was a relationship between DHEAS and SHBG levels (r = 0.51) and between T and SHBG (r = 0.31). We conclude that DHEAS may be a significant modulator of SHBG in the female hirsute patient, an observation seldom mentioned in previous reports.     

Delineation and synthesis of the membrane receptor-binding domain of sex hormone-binding globulin

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein which binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to identify, isolate, sequence, and synthesize the region of SHBG that interacts with its membrane receptor. To accomplish this, highly purified human SHBG was digested with trypsin. The SHBG-derived tryptic peptides were separated by high performance liquid chromatography. They were evaluated for their ability to compete with 125I-SHBG for binding to the SHBG receptor solubilized from human prostatic membranes. Only a single peptide, corresponding to residues 48-57 of the known sequence of human SHBG, inhibited receptor binding. A synthetic decapeptide with this amino acid sequence also competitively inhibited SHBG binding.     

Study of the carbohydrate moiety of human serum sex hormone-binding globulin

Sex hormone-binding globulin from human blood serum contains two biantennary N-linked oligosaccharide chains of the N-acetyllactosamine type and one O-linked oligosaccharide per one molecule of the glycoprotein. These conclusions have been based on the results of methylation analysis of the whole glycoprotein and investigation of the structures of its glycopeptides prepared using pronase digestion.     

Immunohistochemical localization of sex hormone-binding globulin in normal and neoplastic breast tissue.

The distribution of sex hormone-binding globulin-like antigens (SHBG-LA) in normal and neoplastic human breast tissues was investigated by immunohistochemistry, employing a monospecific polyclonal antiserum against highly purified human SHBG and an avidin-biotin-peroxidase complex (ABC) method in formalin-fixed paraffin embedded tissue sections. In normal breast tissues the staining of SHBG-LA was present exclusively in the cytoplasm of epithelial cells of ductal and ductular types. Nuclei as well as stromal and lymphatic tissues remained unstained. While the staining was positive in all cases of intraductal carcinoma, only 4 out of 15 infiltrating carcinomas revealed SHBG-LA. The demonstration of a plasma sex steroid binding globulin in the cytoplasm of endocrine target cells is consistent with the hypothesis that steroid-binding globulins are able to enter target cells. The apparent loss of this specific cell function in infiltrating carcinomas may result from dedifferentiation and change of cell membrane properties occurring during the process of neoplastic progression.     

Biological function of the carbohydrate component of human sex hormone-binding globulin

The role of the carbohydrate component of sex steroid-binding globulin (SBP) from human blood in the glycoprotein interaction with the recognition system for SBP-estrogen complexes in human decidual endometrium plasma membrane was studied. It was shown that the removal of N-acetylneuraminic acid residues from the oligosaccharide chains of SBP did not affect the steroid-binding or immunochemical properties of the glycoprotein. At the same time, the above modification of the glycoprotein resulted in a loss by SBP of its ability to specifically interact with the membrane recognition system. It is concluded that the oligosaccharide chains of SBP are involved in the formation of determinants needed for recognition of the SBP-estrogen complexes by endometrium cell plasma membranes.     

Low polarity ligands of sex hormone-binding globulin in pregnancy. Part II--Identification

Certain previously unrecognized ligands of SHBG of low polarity in pregnancy were identified. They include two weakly bound compounds: 5 alpha-pregnane-3,20-dione and progesterone; and two strongly bound substances, 2-methoxyestrone and a new steroid, estradienolone (17 beta-hydroxy-1,5-estradiene-3-one). The identification of the first three peaks was based on chromatographic elution patterns, binding characteristics and gas chromatography-mass spectrometry. The identification of the fourth peak, the new steroid, was based on similar kinds of evidence and, in addition, solubility characteristics and ultraviolet absorption spectrum.