Geoffrey L. Hammond

The serum transport of steroid hormones.

Publisher Summary This chapter discusses the serum transport of steroid hormones. Steroid hormones are extensively bound to plasma proteins including albumin, corticosteroid binding globulin (CBG), and sex hormone binding globulin (SHBG). Because of its high concentration, albumin binding is important in determining the magnitude of the nonprotein bound or free fraction of a steroid in plasma. The generally accepted model of steroid hormone action suggests that free steroid (in equilibrium with circulating binding proteins) diffuses passively through target cell membranes and binds to a soluble intracellular receptor. The steroid-receptor complex apparently moves into the nucleus where it modifies the chromatin transcriptional activity which results in, among other things, altered levels of protein synthesis. CBG has been differentiated from the intracellular glucocorticoid and progesterone receptors by its inability to bind synthetic glucocorticoids and progestins.

A versatile method for the determination of serum cortisol binding globulin and sex hormone binding globulin binding capacities

Cortisol binding globulin (CBG) and sex hormone binding globulin (SHBG) are plasma glycoproteins which bind some of the most important steroid hormones in blood with high affinity and specificity, but limited capacity. Although antisera have been raised against purified preparations of these proteins and radioimmunoassays have been developed to measure their actual serum concentrations [ 1,2], determinations of their steroid binding capacities will continue to be of importance, especially if variants with abnormal steroid binding activities exist [3,4]. In general, most methods currently used to determine the serum or plasma binding capacities of CBG and SHBG yield values which are in close agreement [5,6]. However, some of these methods are not suited for the routine analysis of large numbers of samples [7,8] while others require the use of expensive gels [9,10], or work under cold room conditions [ 111. Ammonium sulphate precipitation is probably the most widely used method to determine serum SHBG binding capacity [ 12141, but this technique is not so easily applied for measurement of CBG binding capacity, and cannot be readily adapted to measure SHBG binding capacity in samples containing low concentrations of protein, such as obtained during purification procedures. It has been our intention therefore to develop a reliable, inexpensive, versatile method that is suitable for the routine determination of both CBG and SHBG in dilute serum samples.

Potential functions of plasma steroid-binding proteins

The plasma steroid-binding proteins, sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG), transport steroid hormones in the blood and regulate their access to target tissues. Recent biochemical and molecular analyses of these proteins and their genes, and studies of their biosynthesis and localization in the liver and other tissues during development, have led to the realization that CBG and SHBG function in much more sophisticated ways. In particular, the presence of plasma membrane binding sites for both CBG and SHBG on steroid target cells, and evidence for interactions between CBG and specific proteinases at sites of inflammation or tissue remodeling, suggest that these proteins control steroid hormone bioavailability and/or action in a highly selective or targeted fashion. This new information should not only serve to extend our understanding of the basis of steroid-hormone dependent diseases, but may influence the design of steroid hormone agonists and antagonist of therapeutic potential.

Monosaccharide-induced lipogenesis regulates the human hepatic sex hormone–binding globulin gene

The liver produces plasma sex hormone-binding globulin (SHBG), which transports sex steroids and regulates their access to tissues. In overweight children and adults, low plasma SHBG levels are a biomarker of the metabolic syndrome and its associated pathologies. Here, we showed in transgenic mice and HepG2 hepatoblastoma cells that monosaccharides (glucose and fructose) reduce human SHBG production by hepatocytes. This occurred via a downregulation of hepatocyte nuclear factor-4alpha (HNF-4alpha) and replacement of HNF-4alpha by the chicken OVA upstream promoter-transcription factor 1 at a cis-element within the human SHBG promoter, coincident with repression of its transcriptional activity. The dose-dependent reduction of HNF-4alpha levels in HepG2 cells after treatment with glucose or fructose occurred in concert with parallel increases in cellular palmitate levels and could be mimicked by treatment with palmitoyl-CoA. Moreover, inhibition of lipogenesis prevented monosaccharide-induced downregulation of HNF-4alpha and reduced SHBG expression in HepG2 cells. Thus, monosaccharide-induced lipogenesis reduced hepatic HNF-4alpha levels, which in turn attenuated SHBG expression. This provides a biological explanation for why SHBG is a sensitive biomarker of the metabolic syndrome and the metabolic disturbances associated with increased fructose consumption.

Crystal structure of human sex hormone-binding globulin: steroid transport by a laminin G-like domain.

Human sex hormone‐binding globulin (SHBG) transports sex steroids in blood and regulates their access to target tissues. In biological fluids, SHBG exists as a homodimer and each monomer comprises two laminin G‐like domains (G domains). The crystal structure of the N‐terminal G domain of SHBG in complex with 5α‐dihydrotestosterone at 1.55 Å resolution reveals both the architecture of the steroid‐binding site and the quaternary structure of the dimer. We also show that G domains have jellyroll topology and are structurally related to pentraxin. In each SHBG monomer, the steroid intercalates into a hydrophobic pocket within the β‐sheet sandwich. The steroid and a 20 Å distant calcium ion are not located at the dimer interface. Instead, two separate steroid‐binding pockets and calcium‐binding sites exist per dimer. The structure displays intriguing disorder for loop segment Pro130–Arg135. In all other jellyroll proteins, this loop is well ordered. If modelled accordingly, it covers the steroid‐binding site and could thereby regulate access of ligands to the binding pocket.

The cDNA-deduced primary structure of human sex hormone-binding globulin and location of its steroid-binding domain

We have sequenced a cDNA for sex hormone‐binding globulin (SHBG) isolated from a phage λgt 11 human liver cDNA library. The library was screened with a radiolabeled rat androgen‐binding protein (ABP) cDNA, and the abundance of SHBG cDNAs was 1 in 750 000 plaques examined. The largest human SHBG cDNA (1194 base‐pairs) contained a reading frame for 381 amino acids. This comprised 8 amino acids of a signal peptide followed by 373 residues starting with the known NH2‐terminal sequence of human SHBG, and ending with a termination codon. The predicted polypeptide M r of SHBG is 40 509, and sites of attachment of one O‐linked (residue 7) and two N‐linked oligosaccharide (residues 351 and 367) chains were identified. Purified SHBG was photoaffinity‐labeled with Δ6‐[3H]testosterone and cleaved with trypsin. The labeled tryptic fragment was isolated by reverse‐phase HPLC, and its NH2‐terminal sequence was determined. The results suggest that a portion of the steroid‐binding domain of SHBG is located between residue 296 and the 35 predominantly hydrophilic residues at the C‐terminus of the protein.

Genetic Determinants of Serum Testosterone Concentrations in Men

Testosterone concentrations in men are associated with cardiovascular morbidity, osteoporosis, and mortality and are affected by age, smoking, and obesity. Because of serum testosterones high heritability, we performed a meta-analysis of genome-wide association data in 8,938 men from seven cohorts and followed up the genome-wide significant findings in one in silico (n = 871) and two de novo replication cohorts (n = 4,620) to identify genetic loci significantly associated with serum testosterone concentration in men. All these loci were also associated with low serum testosterone concentration defined as <300 ng/dl. Two single-nucleotide polymorphisms at the sex hormone-binding globulin (SHBG) locus (17p13-p12) were identified as independently associated with serum testosterone concentration (rs12150660, p = 1.2×10−41 and rs6258, p = 2.3×10−22). Subjects with ≥3 risk alleles of these variants had 6.5-fold higher risk of having low serum testosterone than subjects with no risk allele. The rs5934505 polymorphism near FAM9B on the X chromosome was also associated with testosterone concentrations (p = 5.6×10−16). The rs6258 polymorphism in exon 4 of SHBG affected SHBGs affinity for binding testosterone and the measured free testosterone fraction (p<0.01). Genetic variants in the SHBG locus and on the X chromosome are associated with a substantial variation in testosterone concentrations and increased risk of low testosterone. rs6258 is the first reported SHBG polymorphism, which affects testosterone binding to SHBG and the free testosterone fraction and could therefore influence the calculation of free testosterone using law-of-mass-action equation.

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. This review compares the production and functions of sex hormone-binding globulin in different species and evaluates the diverse effects this has on reproduction.

Serum steroid binding protein concentrations distribution of progestogens and bioavailability of testosterone during treatment with contraceptives containing desogestrel or levonorgestrel.

The oral administration of 150 micrograms desogestrel and 30 micrograms ethinyl estradiol (EE2) increases (P less than 0.001) serum concentrations of sex-hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG), whereas treatment with 150 micrograms levonorgestrel and 30 micrograms EE2 only increases serum CBG concentrations. No changes in serum albumin concentrations occurred during or after treatment with either preparation, and increases in SHBG and CBG returned to the pretreatment values 1 month after treatment ceased. The serum distribution of levonorgestrel was unchanged during treatment, whereas the increase in serum SHBG concentrations after treatment with the preparation containing desogestrel decreased (P less than 0.001) the percentage of non-protein-bound 3-keto- desogestrel and the percentage of albumin-bound 3-keto- desogestrel but increased (P less than 0.001) the SHBG-bound fraction. Oral contraceptives containing either progestogen decrease the mean serum non-protein-bound testosterone concentrations, especially during treatment with desogestrel (P less than 0.001), and desogestrel may therefore by the more appropriate progestogen for the treatment of women prone to androgenic side effects.

Molecular studies of corticosteroid binding globulin structure, biosynthesis and function

Phylogenetic comparisons of the primary structure of corticosteroid binding globulin (CBG) have revealed several conserved domains that include sites for N-glycosylation and a region which probably represents a portion of the steroid binding site. The major site of CBG biosynthesis in adults is clearly the liver, and the human CBG gene promoter contains sequence elements that interact with liver-specific transcription factors. Low levels of CBG gene expression have been detected in other tissues, and these may be important for fetal development during late gestation when hepatic CBG mRNA levels are low. Studies of the ontogeny of CBG biosynthesis in the rat have also indicated that plasma CBG levels may be influenced by a more rapid clearance of the protein during pubertal development. Analyses of the structural organization and chromosomal location of the human CBG gene have further confirmed its close relationship with the serine proteinase inhibitors, and suggests that CBG, alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin evolved relatively recently by gene duplication. The functional significance of this relationship has been examined and our studies suggest that a specific interaction between CBG and elastase on the surface of neutrophils may represent a physiologically important event that promotes the delivery of glucocorticoids to these cells at sites of inflammation.