Specific steroid-binding glycoproteins of human blood plasma: novel data on their structure and function

In this review, the modern data on the polypeptide and carbohydrate structures of human corticosteroid-binding globulin (CBG) and sex hormone-binding globulin (SHBG) as well as on the biochemical properties and biological functions of these steroid-binding glycoproteins are discussed.     

Relative binding affinity of norgestimate and other progestins for human sex hormone-binding globulin

The relative binding affinity of norgestimate for human sex hormone-binding globulin was compared with that of its metabolites and other progestins by measuring their abilities to displace [3H]testosterone from this carrier protein in vitro. Norgestimate and its 17-deacetylated and 3-keto metabolites did not significantly displace [3H]testosterone from sex hormone-binding globulin at concentrations up to 10,000 nM, whereas gestodene, levonorgestrel, and 3-keto desogestrel displaced [3H]testosterone from sex hormone-binding globulin with IC50 concentrations of 23.1, 53.4, and 91.0 nM, respectively. Since it is believed that a progestin may exert androgenic effects by displacing testosterone from sex hormone-binding globulin, thereby increasing circulating levels of free, active testosterone, these data are consistent with the results of preclinical and clinical studies demonstrating the selective progestational activity of norgestimate.     

Sex hormone-binding globulin selectively modulates estradiol-regulated genes in MCF-7 cells.

Human sex hormone-binding globulin inhibits the effects of estradiol on proliferation and apoptosis of breast cancer cells. We report here the effect of sex hormone-binding globulin on estradiol regulation of gene expression in MCF-7 breast cancer cells using a selected set of genes. Estradiol upregulates genes that are positive regulators of proliferation (e.g., bcl-2, c-fos, c-myc, cyclin D) or/and related to more aggressive form of breast cancer (e.g. BRCA-1, EGF-R) and downregulates two genes (c-jun and ERalpha). Sex hormone-binding globulin modulates only a selected group of estradiol-controlled genes (inhibiting upregulation of bcl-2, c-myc, EGF-R, PR, and downregulation of ERalpha), starting 48 hours after treatment. Our study demonstrates that in breast cancer cells, sex hormone-binding globulin is effective on few selected genes which are involved in cell growth and apoptosis or related to cell estrogen-dependence and that the protein regulation of estradiol effect is selected and specific. Sex hormone-binding globulin action in estrogen breast cancer cells is strongly associated to cell growth and estrogen-sensitivity.     

Binding of phytoestrogens to rat uterine estrogen receptors and human sex hormone-binding globulins

The interaction of phytoestrogens with the most important binding sites of steroid hormones, i.e. sex hormone-binding globulin and estrogen receptors, was investigated. Relative binding affinities and association constants for 21 compounds among them isoflavones, flavones, flavonols, flavanones, chalcones and lignans were determined. The lignan nordihydroguaiaretic acid weakly displaced 17beta-[3H]-estradiol from estrogen receptor and Scatchard analysis suggests non-conformational changes. Compounds from Glycyrrhiza glabra, liquiritigenin and isoliquiritigenin, showed estrogenic affinities to both receptors. 18beta-Glycyrrhetinic acid displaced 17beta-[3H]-estradiol from sex hormone-binding globulin but not from the estrogen receptor. Phytoestrogens compete with 17beta-estradiol much stronger than with 5alpha-dihydrotestosterone for binding to sex hormone-binding globulin.     

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.     

Interrelationships of serum testosterone and free testosterone index with FFM and strength in aging men

Muscle mass and strength losses during aging may be associated with declining levels of serum testosterone (T) in men. Few studies have shown a direct relationship between T and muscle mass and strength. Subjects were 262 men, aged 24-90 yr, from the Baltimore Longitudinal Study of Aging, who had T and sex hormone-binding globulin sex hormone-binding globulin (SHBG) measurements, from which the free T index (FTI) was calculated (T/SHBG) from serum samples collected longitudinally since 1963, total body fat mass and arm and leg fat-free mass (FFM) by dual-energy X-ray absorptiometry and arm and leg strength by dynanomometry. Mixed-effects models estimated T and FTI at the time of mass and strength measurements. Age, total body fat, arm and leg FFM, T, and FTI were significantly associated with concentric and eccentric strength. FTI, not T, was modestly, but directly, related to arm and leg strength after fat, arm and leg FFM, height, and age were accounted for and indirectly through body mass. FTI is a better predictor of arm and leg strength than T in aging men.     

Are estrogen receptor content in breast cancer and effects of tamoxifen on sex hormone-binding globulin markers for individual estrogen sensitivity?

Individual women differ with respect to their sensitivity to estrogen and serum levels of sex hormone-binding globulin (SHBG) may reflect the individual response. We found a significant correlation between estrogen receptor (ER) concentrations in breast cancer tissue and SHBG levels during tamoxifen treatment. Estrogen sensitivity may be a general characteristic common to various organs and different between individual women.     

Steroid-binding proteins in follicular fluid and peripheral plasma from pigs, cows and sheep

No unusual steroid-binding proteins that might react with the oocyte or its investments could be detected in follicular fluid. Corticosteroid-binding globulin occurred in follicular fluid from pigs, sheep and cows, and sex hormone-binding globulin occurred in follicular fluid from sheep and cows. The bulk of the steroid in follicular fluid is bound to albumin with low affinity, indicating that steroid molecules can readily be released, and oestrogen can react with the oocyte and granulosa cells in a manner analogous to that demonstrated for target cells bathed with interstitial fluid. Pigs lack a sex-hormone binding globulin in blood plasma and, hence, in follicular fluid. Because no proteins exist in follicular fluid that would compete with antibodies to bind steroids, direct radioimmunoassay of follicular steroids appears to be a valid technique.     

Localization of the human sex hormone-binding globulin gene (SHBG) to the short arm of chromosome 17 (17p12----p13)

Human sex hormone-binding globulin (SHBG) is a plasma steroid transport protein which is known to be encoded by an autosomal gene. We have hybridized two separate cDNA probes, corresponding to the 5' and 3' portions of the coding sequence for SHBG, to human metaphase chromosomes in situ. In this way, the SHBG gene has been localized to the p12----p13 bands of chromosome 17.     

Estradiol induction of cAMP in breast cancer cells is mediated by foetal calf serum (FCS) and sex hormone-binding globulin (SHBG).

Plasma sex hormone-binding globulin (SHBG or SBP), the specific carrier for estradiol and androgens, after binding to its membrane receptor (SHBG-R), causes a significant increase of cAMP in the presence of estradiol, in both breast (MCF-7) and prostate (LNCaP) cancer cells maintained in serum-free medium. On the other hand, it has been proposed that estrogens, in addition to the well-known nuclear receptor pathway, exert their biological effect inducing cAMP, as a consequence of a direct membrane action, in breast cancer and uterine cells. The aim of the present study was to clarify this controversial issue by verifying if the cAMP increase in MCF-7 cells was a direct effect of estradiol, or if it was mediated by FCS proteins, such as bovine sex hormone-binding globulin; and to reevaluate the effect of human SHBG on cAMP induction in the presence of FCS. MCF-7 cells were maintained in DCC-FCS (treated with DCC to remove steroids), in SHBG-FREE/DCC-FCS (treated with DCC and with a specific affinity chromatography to remove bovine sex hormone-binding globulin), or in serum-free medium (SFM). It was observed that estradiol determined a significant time-dependent increase of cAMP only in MCF-7 cells maintained in 10% DCC-FCS. When cells were maintained in 10% SHBG-FREE/DCC-FCS, estradiol had no detectable effect. However, its ability to increase cAMP was observed again after the addition of human SHBG, in doses ranging from 5 to 50 nM. Moreover, in the presence of 10% SHBG-FREE/DCC-FCS, SHBG, even in the absence of estradiol, caused a significant increase of cAMP. In conclusion, the data reported in the present study suggest that the ability of estradiol to induce cAMP in MCF-7 cells is not due to a direct membrane effect of the hormone, but rather it is mediated by FCS. SHBG is one of the serum factors mediating estradiol action. Lastly, it was proven that SHBG triggers the cAMP pathway in MCF-7 cells in a physiologic culture condition and at physiologic concentrations.     

Thyroid hormone generalized resistance.

The syndromes of thyroid hormone resistance may affect overall or only some tissues. The generalized resistance is an inherited disease which involves a familial eumetabolic or hypometabolic goiter, increased free thyroid hormones with normal or elevated plasma TSH levels; children may present mental retardation, deafness, short stature and delayed bone age. The disease is frequently misdiagnosed. In vivo and in vitro tests may be used to assess the diagnosis. The defect of increment of sex hormone-binding globulin after administration of T3 may be useful in the demonstration of the disease. Therapy uses high T4 or T3 doses in hypometabolic patients. The generalized thyroid hormone resistance could be linked to abnormalities at the T3 receptor and c-erb A gene level, as a consequence of different point mutations or deletions involving the hormone-binding domain.     

A method of purification of partially methylated alditol acetates in the methylation analysis of glycoproteins and glycopeptides

A method for methylation analysis of intact glycoproteins is described. Starting with intact glycoprotein, the oligosaccharides are methylated, hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates are then separated from noncarbohydrate contaminants on a silica gel G column. Partially methylated hexitol acetates are eluted from the column with petroleum ether:ethyl acetate (1:1, $\text{v}\text{v}$) and partially methylated N-acetylhexosaminitol acetates are subsequently eluted with methanol. Analysis by gas-liquid chromatography/mass spectrometry of the partially methylated alditol acetates shows no interfering contaminants. This method circumvents the need to make pronase glycopeptides and avoids the pitfalls of other methylation procedures.     

Androgènes et sexualité masculine

Testosteron is known to be critical for the right maintenance of masculin sexuality. It acts on all components of sexuality: libido, erection and ejaculation. As far as erection is concerned, one has to emphasize on the fact that only spontaneous nocturnal erections are androgen-dependent unlike erectile responses to visual erotic stimuliThe T can act, at the level of target organes, either as unchanged or as more often observed after reduction to DHT or aromatisation to E2. It is essencially as DHT that the male hormone seems to act on the sexuality at both the central and peripherical levels. The oestrogens even if now shown to have no peripherical action, are still subject to controversy as far as their central action is concerned. Therefore an action at the cerebral level of the oestrogens produced locally can not be definitively eliminated.The concentrations of T required for the re-establishment of the sexual function in case of hypogonadism are closer to the lower limits of normal values. The upper treshold of the action of T on the sexuality, if any, would be at supra-physiological levels.Androgenotherapy of sexual dysfonctionnalities is wittnissing a constant evolution. New and more performing products, as well as novel and less astreignant ways of administration, are already disponible or under evaluation. Moreover, the improvement of plasmic androgen exploration (dosage of free and non-sex hormone-binding globulin bound testosterone) associated to a better understanding of side effects of the androgenic therapeutics may allow in the near futur to reach a consensus on the androgenotherapy indications. As a matter of fact among the population of patients considered eugonadic (based on normal levels of T) and showing a sexual dysfonctionality, two subgroups might be interested: That of elderly patients displaying either an increase of the hormone-binding globulin (SHBG) or a decrease of the non-sex hormone-binding globulin bound testosterone levels and that of young patients suffering from idiopathic impotence. It should be, however, pointed at that the recent character of the different physiopathologic and therapeutic acquisitions requires a validation through larger series and long term studies.     

Growth and anabolic hormones,leptin, and neuromuscular performance in moderately trained prepubescent athletes and untrained boys

We investigated hormonal regulators of growth and development, leptin levels, body composition, neuromuscular performance, and the associations among them in trained prepubertal athletes (experimental group [EG]) and an untrained control group (CG). Informed consent was obtained from the children and their parents. Their maturation stage was evaluated according to Tanner's criteria. There were no differences between EG and CG in physical characteristics, body mass index (BMI), lean body mass, testosterone (T), sex hormone-binding globulin, free androgen index, growth hormone (GH), hand grip strength, and jumping performance. Leptin levels and percent fat of the EG were significantly lower than those of the CG (p < 0.05-0.005). Leptin levels were significantly correlated to body fat and BMI for both the EG and the CG (r = 0.51-0.79). There is little evidence that leptin has a positive effect on growth and anabolic factors. Sex hormone-binding globulin and GH may explain the variation of leptin in athletes with low T (R(2) = 0.43) and in CG (R(2) = 0.35), respectively. Leptin seems to be a permissive factor for the onset of puberty, and the training background needs an optimal biological maturation to produce significant differences in muscle and power performance.     

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 diet on plasma steroids and sex hormone-binding globulin levels in adult men

Several experimental studies have suggested that diet can alter the production and metabolism of steroids in men. The purpose of this study was to determine the levels of unconjugated steroids and steroid glucuronides as well as sex hormone-binding globulin (SHBG) among normal adult men who were either omnivorous or vegetarians. The participants were white volunteers ranging from 25-35 years of age and the blood samples were taken between 0900 h and 1000 h and between 1600 h and 1700 h for two consecutive days. No significant statistical change was found in plasma dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, dihydrotestosterone and estradiol levels. Vegetarian group showed a higher levels of sex hormone-binding globulin (SHBG) while the free androgen index (FAI; calculated by the ratio testosterone/SHBG) was lower in this group. Although the concentrations of androsterone glucuronide were higher in vegetarian group, the vegetarians had a 25-50% lower level of androstane-3 alpha, 17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide. Our data further indicate that both, androstane-3 alpha,17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide concentrations are significantly correlated with SHBG levels and with the FAI values. The increases in androstane-3 alpha,17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide levels in the omnivorous group are probably a consequence of the elevation of the FAI. Our data suggest that in a vegetarian group, less testosterone is available for androgenic action.     

A simple enzyme-linked immunosorbent assay for sex hormone-binding globulin

A simple enzyme-linked immunosorbent assay (ELISA) for sex hormone-binding globulin (SHBG) has been developed. Polyclonal antibody raised to SHBG purified to homogeneity was employed. The ELISA, which may be performed in under 4 h, shows no cross-reactivity with other serum proteins, has a sensitivity of less than 1.2 fmol per sample, demonstrates excellent correlation with ligand-binding techniques (r = 0.996; p less than 0.0001), and has intra- and inter-assay coefficients of variation of between 5-9% and 7-11% respectively.     

Estradiol,progesterone, and sex hormone-binding globulin in female rhesus monkeys (Macaca mulatta)

We measured the concentrations of estradiol, progesterone, and the sex hormone-binding globulin capacity (rhSHBG) in serum of female rhesus monkeys (Macaca mulatta). Although the serum rhSHBG capacity was altered by the removal of ovarian hormones, presumably estradiol, acute changes in serum estradiol and progesterone did not influence SHBG capacity. There appears to be a relatively low threshold for the effect of estradiol on rhSHBG capacity. The threshold must be present for a finite length of time to have that effect.     

The metabolism of human sex hormone-binding globulin in the rhesus monkey.

The metabolism of human sex hormone-binding globulin (hSHBG) was studied in eight female rhesus monkeys (Macaca mulatta) after the pulse injection of [125I]-hSHBG. hSHBG was iodinated with 125I using a chloramine T technique, and the [125I]-hSHBG was separated from other constituents by molecular sieve chromatography with a Sephadex G-25 column. The [125I]-hSHBG was administered intravenously as a pulse in 2 ml of phosphate buffer, pH 7.4, to each of eight rhesus monkeys. Blood samples (2.0 ml) were obtained at 2, 4, 6, 8, 24, 30, 45, and 54 hr after the injection. The glycoproteins were precipitated with concanavalin A-Sepharose, and the radioactivity was measured. The concentration of radioactivity as fraction of dose/ml of serum was plotted on a semilog scale against time. The disappearance of radioactivity could be expressed best as the sum of two exponentials, with a mean +/- SE t1/2 of 2.5 +/- 0.4 and 33.1 +/- 3.7 hr, respectively. The initial volume of distribution was 461 +/- 78 ml and the metabolic clearance rate was 559 +/- 66 ml/day. The very low clearance rate and prolonged t1/2 are compatible with a relative stability in the circulating mass of SHBG. Rapid changes in concentration of SHBG could be due to changes in serum volume, reversible changes in tissue distribution of SHBG, or the secretion of variable forms of desialylated SHBG.     

Genetic mapping of the gene for androgen-binding protein/sex hormone-binding globulin to mouse chromosome 11

Southern blot analysis of DNAs from Chinese hamster x mouse and rat x mouse somatic cell hybrids showed that the mouse gene encoding androgen-binding protein/sex hormone-binding globulin (ABP-SHBG) is on Chromosome 11. Progeny from an intersubspecies backcross were analyzed to position this locus, termed Shbg, between Il-3 and Int-4 in the middle of this chromosome. Shbg is thus closely linked to several neurological mutations, one of which, Tr, is also associated with male sterility. The recent finding that ABP-SHBG is found throughout the rat brain raises the possibility that one of these mutations may be due to a defect in Shbg.