Effects of sex hormone binding globulin (SHBG) on human prostatic carcinoma

The purpose of this study was to determine what effects sex hormone binding globulin (SHBG) might have on the growth and steroid content of human prostate carcinoma. Two human prostate carcinoma cell lines were used for this study, ALVA-41 and ALVA-101. The first part of the study was to determine the effect of SHBG or albumin on the uptake of [³H]DHT in the cells. In this experiment both SHBG and albumin inhibits the uptake of [³H]DHT into each of the cell lines when studied in vitro. The degree of inhibition was dependent on the binding capacity of the protein. When [³H]thymidine uptake was measured in each of the cell lines following either the addition of SHBG or albumin to the culture media, an increase in uptake and presumably DNA synthesis was noted in the ALVA-41 and ALVA-101 cells for SHBG additions but not for albumin. Further, this stimulation was increased when testosterone was added to the media, however, [³H]thymidine uptake was decreased by high concentrations of dihydrotestosterone (DHT) or if the SHBG was saturated with DHT prior to being added to the media. The cells also demonstrate high affinity cell membrane receptors for SHBG. Finally, using a 3′, 550 bp cDNA or SHBG, 1.9 and 2.8 kb mRNAs were detected on Northern analysis of the ALVA-101 and ALVA-41 cells. These data indicate SHBG can inhibit uptake of steroids into the prostrate, but also it may act as a stimulus for growth through a SHBG cell surface receptor. In addition, the growth effect may be through an autocrine effect from SHBG or a SHBG-related peptide.     

A liquid-phase immunoradiometric assay (IRMA) for human sex hormone binding globulin (SHBG)

An immunoradiometric assay (IRMA) for sex hormone binding globulin (SHBG) has been developed in which an 125I-labeled monoclonal antibody [( 125I]S1B5) and a rabbit anti-SHBG antiserum (RAb) are incubated in "liquid-phase" with standards or samples, and RAb-bound complexes are separated using donkey anti-rabbit IgG antibody-coated cellulose. This immunoassay technique is characterized by several advantages; the [125I]S1B5 imparts additional specificity and obviates the requirement for pure SHBG; the use of excess reagents reduces incubation times and also improves assay performance and sensitivity, and incubation in "liquid-phase" conserves and increases the efficiency of the RAb. The assay measures only non-denatured SHBG and is not influenced by the presence of steroid at the binding site. Assay specificity was demonstrated by parallelism between dilutions of pure SHBG and different serum samples. The quantitative recovery of SHBG added to serum, and the agreement between specific activities of SHBG in pure standards and sera, confirm the accuracy of the method. The within and between assay coefficients of variation were less than 7% and less than 11%, respectively, between 12 and 450 nmol/l. The assay sensitivity may be manipulated by altering the concentration of RAb and/or by preincubation with either [125I]S1B5 or RAb, and 0.2 fmol SHBG may be measured on a standard curve. The SHBG assay has been used to measure SHBG concentrations in sera, amniotic fluid, cerebral spinal fluid, seminal plasma and saliva.     

2-Iodoestradiol binds with high affinity to human sex hormone binding globulin (SHBG)

Human sex hormone binding globulin (SHBG) binds a set of steroids that differ slightly from each other in structure. Dihydrotestosterone and testosterone are bound with high affinity by SHBG whereas estradiol is bound with a lower affinity. In this work we have studied the binding to human SHBG of the derivatives obtained by substituting iodine in the aromatic A-ring of estradiol. Three A-ring iodinated estradiol derivatives, 2-iodoestradiol, 4-iodoestradiol and 2,4-di-iodoestradiol, were obtained by treating 17 beta-estradiol with NaI and Chloramine T and separating the reaction products by HPLC. Their structures were confirmed by mass spectrometry and 1H-NMR. The corresponding radioactive compounds were obtained with use of Na[125I] in the same synthesizing procedure. Incubation of whole serum, serum albumin and purified SHBG with each of the three [125I]iodoestradiols followed by agarose gel electrophoresis showed only 2-iodoestradiol to have a strong binding to SHBG. This steroid was also bound to albumin, but with a lower affinity. Besides SHBG and albumin, there were no other binders of 2-iodoestradiol in human serum. The affinity constant for the binding of 2-iodoestradiol to purified human SHBG at 37 degrees C and physiological pH was determined by a dextran-coated charcoal method to be 2.4 x 10(9) M-1 (i.e. exceeding that of dihydrotestosterone). It was found that 0.9 mol of 2-iodoestradiol was bound per mol of SHBG dimer (93 kDa) at saturation, and that 2-iodoestradiol competed with dihydrotestosterone for the same binding site of SHBG. It was concluded that 2-iodoestradiol has a remarkably high affinity for human SHBG, and that its gamma-emitting 125I-analog is useful for binding studies of human SHBG.     

Pathophysiology of sex hormone binding globulin (SHBG): Relation to insulin

In humans, the plasma level of sex hormone binding globulin (SHBG) is regulated by several hormones. We have now accumulated evidence that SHBG is also intimately related to nutritional state. However, we do not yet know what specific signal, if any, may be the regulator of SHBG.

There is a strong and negative correlation between fasting insulin level and SHBG in obese as in hyperandrogenic women. Under such circumstances, a high fasting insulin level, normal glycemia and a low SHBG level suggest insulin resistance in terms of glucose disposal but not in terms of SHBG inhibition. This is a rather complex situation.

It is too early to judge the importance of IGF-I in the regulation of SHBG. But it may turn out that IGF-I is the main regulator of SHBG and that, by interaction with the IGF-I receptors, insulin carries on its inhibitory activity on SHBG.     

The effects of sex hormone binding globulin (SHBG) on testosterone transport into the cerebrospinal fluid.

The movement of testosterone (T) from blood across the blood-brain barrier (BBB) is thought to reflect the combined effects of T's lipid solubility and the presence of circulating binding proteins for T such as albumin or sex hormone binding globulin (SHBG). Since the adult rat lacks a circulating specific high affinity sex steroid binding protein, examination of the disappearance from serum and uptake into cerebrospinal fluid (CSF) of [3H]T before and after SHBG or albumin infusion should provide insight into the function of these two proteins with respect T transport. Three groups of adult male Sprague-Dawley rats were cannulated at the femoral vein and cisterna magna. In a control group (n = 8), [3H]T was given as an intravenous bolus beginning at time zero; multiple serum and CSF collections were assayed for counts per min (cpm) during the subsequent 45 min. Data from these animals were then compared to those seen in animals that received either purified human SHBG (hSHBG) (n = 7) or human albumin (hALB) (n = 6) 10 min prior to the [3H]T infusion. High performance liquid chromatography was used to monitor the metabolic fate of the steroid infusate at the end of each study period. Infusion of hSHBG increased serum concentrations from undetectable to 93.8 nM/l (mean +/- SEM, n = 6). Administration of hALB significantly increased (25.0 +/- 1.2 g/l at baseline, 33.4 +/- 1.6 g/l post-infusion, mean +/- SEM, P less than 0.03, n = 5) the circulating albumin concentration. Comparison of data from each group of animals demonstrated that (1) following an i.v. injection of radiolabeled T, the initial decline in serum [3H]T was significantly reduced (P less than 0.03) in the presence of hSHBG, (2) hALB did not affect the movement of [3H]T out of serum, (3) the time to peak appearance of [3H]T in the CSF was significantly delayed (P less than 0.02) by the presence of circulating hSHBG, and (4) the net quantity of [3H]T found in the cSF under steady-state conditions was not affected by serum SHBG or albumin levels. This study demonstrates that high-affinity steroid binding proteins do modulate the transport of sex steroids across the BBB. Specifically, SHBG delays the clearance of T from serum and slows the rate of T uptake into the CSF during non-equilibrium conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of a monoclonal antibody to human sex hormone binding globulin

The occurrence and distribution of PHI-like immunoreactivity in the guinea pig gallbladder has been analysed by radioimmunoassay and immunocytochemistry. Chromatography of gallbladder extracts by gel permeation and high-performance liquid chromatography revealed that guinea pig PHI-like immunoreactivity is of a similar size to that of porcine PHI but may differ in its amino acid sequence. Immunocytochemistry showed PHI-immunoreactivity to be localised to nerves found predominantly in the ganglionated plexus and the mucosal plexus of the gallbladder. Pure natural porcine PHI induced a dose-dependent relaxation of the isolated guinea pig gallbladder muscle which was not blocked by antagonists to acetylcholine, catecholamines, histamine, and 5-hydroxytryptamine. PHI may thus be one of the local factors involved in controlling gallbladder function.     

The temperature dependence of the binding of 5 alpha-dihydrotestosterone, testosterone and estradiol to the sex hormone globulin (SHBG) of human plasma

The binding of 5 alpha-dihydrotestosterone (DHT), testosterone and estradiol to the sex hormone binding globulin (SHBG) and albumin in human plasma has been studied at 4, 20 and 37 degrees C using the method of equilibrium partition in an aqueous two-phase system based on dextran, poly(ethylene glycol) and water. The intrinsic association constants for the binding to SHBG and the apparent association constant for the binding to albumin have been determined from Scatchard-type binding plots. The affinity of SHBG for DHT is 1.2-1.3 times higher than that for testosterone and 4 times higher than that for estradiol. The affinity of SHBG for the steroids decreases with increasing temperature. The mean values of the free energy of binding, delta G degree, in the temperature range used are -52.3, -51.7 and -48.9 kJ X mol-1 for the binding of DHT, testosterone and estradiol, respectively, to SHBG. The corresponding values of the enthalpy change, delta H degree, are 73.7, 70.0 and 99.0 J X mol-1 X K-1. These values are discussed in terms of the difference in the structure of the steroids. The affinity of albumin for testosterone and estradiol is almost equal and is lower than that for DHT. The delta G degree for the binding to albumin is about 55% lower than that for the binding to SHBG.     

Direct effect of plasma sex hormone binding globulin (SHBG) on the metabolic clearance rate of 17 beta-estradiol in the primate

Sex hormone binding globulin (SHBG) has been shown to be a major determinant of testosterone clearance in the primate. It has also been suggested that SHBG would also be a determinant of estradiol clearance (MCR-E2). However, published studies have suggested that the MCR-E2 do not always vary with changes in the level of SHBG. Therefore, the present study was undertaken to address this issue. The baseline MCR-E2 was determined in adult male pigtail macaques (Macaca nemestrina). Following the baseline determination of MCR-E2 the animals were infused with either purified human (h) SHBG or antibody against hSHBG, which also has a high degree of cross-reactivity with primate SHBG. Following the infusions of either hSHBG or anti-SHBG, MCR-E2 was again determined. In addition, luteinizing hormone (LH) was measured using a mouse Leydig cell bioassay. Following the infusion of hSHBG, a marked increase in serum SHBG was noted and the MCR-E2 decreased. Associated with the increase in SHBG, the SHBG bound T levels decreased and LH increased. Following the infusion of antibody, serum SHBG decreased, and the MCR-E2 also decreased. With the decrease in SHBG following the antibody infusion, non-SHBG bound T increased and serum LH fell. This study demonstrates that an increase in the serum SHBG levels is associated with a decrease in MCR-E2, however, an acute decrease in serum SHBG also decreases the MCR-E2. This later result demonstrates that factors in addition to serum SHBG binding may be important in determining the MCR-E2.     

Detection of a MspI restriction fragment length polymorphism for the human sex hormone-binding globulin (SHBG) gene

A rare polymorphism in the human sex hormone binding globulin (SHBG) gene was detected using a human SHBG cDNA probe. It is the first DNA sequence variation reported in this gene.     

Immunolocalization of sex hormone-binding globulin (SHBG) in human ovarian follicles and corpus luteum

Sex hormone-binding globulin (SHBG), a hepatic carrier protein for sex steroids is expressed in different steroid-sensitive tissues, including Sertoli cells of the testis. It has been suggested that this protein may be one of the local regulators of spermatogenesis. The expression of SHBG in the ovary is currently unknown. We have previously demonstrated the synthesis of SHBG in granulosa-lutein cells from patients undergoing in vitro fertilization. In this study, the presence of SHBG in human ovarian follicles and corpora lutea is investigated, using immunohistochemistry on adult and fetal ovarian tissue sections. SHBG is localized in the whole granulosa layer at all stages of folliculogenesis, whereas, only isolated theca cells are immunostained. In primordial and primary follicles, the oocyte cytoplasm shows an intense immunostaining, which disappears after the secondary stage. In the microenvironment of the mature oocytes, SHBG is present in the surrounding cumulus cells, the perivitelline space, and nearby the oolemma. In the corpus luteum, SHBG is localized in large luteal cells, whereas, small luteal cells do not show any significant staining. By analogy with the testis, these results raise the question of an involvement of SHBG in the regulation of follicular maturation as well as in luteal function.     

Effect of dietary components, including lignans and phytoestrogens, on enterohepatic circulation and liver metabolism of estrogens and on sex hormone binding globulin (SHBG)

A brief account of our present knowledge on the enterohepatic metabolism of estrogens and on the origin, metabolism and biological effects of mammalian lignans and phytoestrogens is undertaken. Furthermore, recently published results on the effects of dietary fiber, fat and carbohydrates on estrogen metabolism are reviewed. New preliminary results are presented on quantitative assays of lignans and phytoestrogens in urine of women belonging to various dietary and population groups and in a group of chimpanzees. The highest values of lignans and phytoestrogens were found in the non-human primates, and in macrobiotic, lactovegetarian and Japanese women, all groups considered having a low risk for the development of breast and other hormone-dependent cancer. New results on correlations between intake of various fibers, lignan and phytoestrogen excretion and plasma levels of estrogens, free testosterone and SHBG in women are presented. There is a significant positive correlation between the intake of fiber and urinary excretion of lignans and phytoestrogens, and the concentration of plasma SHBG. Fiber intake and urinary excretion of lignans and equol correlated negatively with plasma percentage free estradiol. Enterolactone excretion correlated negatively with plasma free testosterone. It is concluded that dietary macro- and micronutrients seem to play an important role in estrogen metabolism.     

Physicochemical characteristics of human sex hormone binding globulin: evidence for two identical subunits

We have developed a rapid protocol for the purification of human sex hormone binding globulin (SHBG) which allows the protein to be purified from pregnancy serum within 48 h. This minimizes any possible degradation of the protein by serum proteases, and has enabled us to re-examine some important and controversial aspects of its structural composition. Our physicochemical data are consistent with the hypothesis that SHBG is a dimeric glycoprotein composed of 2 protomers that exhibit size heterogeneity (approximately 50 and approximately 52 K daltons). The dimeric SHBG molecule appears to contain only approximately 8% carbohydrate, and sequence information indicates that an N-linked oligosaccharide chain may be attached to residue 7 (asparagine) from the NH2-terminal amino acid (leucine). When compared with earlier reports, differences in the relative amounts of heavy (approximately 52 K) and light (approximately 50 K) protomers, and the microheterogeneity of NH2-terminal amino acids, have led us to conclude that they may be caused by proteolytic degradation in vivo as well as during the storage of blood samples prior to protein purification. However, the NH2-terminal amino acid sequence data indicate that the primary structures of the heavy protomers, which evidently interact to form the majority of SHBG dimer in serum, are similar and may even be identical. Evidence to support this is provided by the observation that a monoclonal antibody, which recognises a configurational epitope, interacts with two epitopes per native dimeric form of human SHBG.     

Profiles of ligands of sex hormone binding globulin in human serum

Ligands of the sex hormone-binding globulin (SHBG) in samples of human serum were extracted into diethyl ether and the dried extracts chromatographed using Sephadex LH-20 chromatography. The resulting fractions were assayed by competitive binding to SHBG against a testosterone standard. Values for dihydrotestosterone and testosterone were similar to those obtained using radioimmunoassay. While the bulk of the material in male and non-pregnant female serum corresponded to other known ligands (5-androstane-3 alpha,17 beta-diol and 5-androstene-3 beta,17 beta-diol), the quantities of material in the androstanediol and androstenediol regions exceeded the known values for these steroids in hirsute women and in late pregnancy, suggesting the presence of other steroids as well. In addition, there was a large amount of material of low polarity present in pregnancy which was not accounted for by recognized circulating ligands. A normal pattern was found in a man with Addison's disease, suggesting that the bulk of SHBG ligands in men are derived from the testis. This was also indicated by the 60-fold higher levels of testosterone and androstenediol seen in normal testicular vein serum. High values of testosterone, androstanediol and androstenediol in a woman with untreated 21-hydroxylase deficiency suggested that large amounts of these compounds (or their precursors) can be produced by the adrenal and that their production by the adrenal is regulated at least in part by ACTH.     

Estrogen and androgen regulation of sex hormone binding globulin secretion by a human liver cell line

Both estrogens and androgens have been shown to stimulate sex hormone binding globulin (SHBG) secretion in vitro in the hepatocellular carcinoma cell line, Hep G2, in contrast to the expected inhibition by androgens from in vivo studies. However, such in vitro stimulation was only demonstrated at high steroid doses, generally in serum-containing medium, with added Phenol Red. In the present study, Hep G2 cells were grown in serum-free medium, without Phenol Red, under the influence of testosterone (T) (0, 0.5-500 nM) and ethinyl estradiol (EE2) (0, 50 pM-500 nM). Levels of secreted SHBG and albumin were correlated with androgen receptors in cytosolic (ARc) and nuclear (ARn) fractions and with DNA levels. In the presence of increasing T levels, SHBG levels fell to 39% of control values at 5 nM T (P = 0.047), rising to 97% of control at 500 nM. Conversely, incubation with EE2 produced a rise in SHBG secretion of more than 100% at 0.5 nM (P less than 0.02) which was sustained to 50 nM (P less than 0.005). DNA levels did not change with the addition of testosterone or EE2, with the exception of a 15% reduction at 5 nM EE2 (P less than 0.05). Albumin levels in the medium were not significantly altered by either steroid. However, in response to T, androgen receptor (AR) levels were reduced in cytosolic (42% of control) and nuclear (22%) fractions at 5 nM, and these changes in ARc and ARn correlated with SHBG levels over the range of T concentrations (P = 0.04 and P = 0.017, respectively). Nuclear estrogen receptor (ER) increased over 10-fold at 5 and 50 pM EE2 (P less than 0.001) and maintained 50 nM (P less than 0.001). Cytosolic ER was reduced at 0.5 and 5 nM but recovered at 50 nM, correlating with SHBG levels (P less than 0.001). These findings are consistent with the hypothesis that estrogens and androgens regulate SHBG synthesis in man by direct, specific, probably receptor-mediated effects on hepatocytes. Hep G2 cells grown in serum-free medium are a suitable experimental system for further study of this phenomenon.