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Testosterone Replacement, Low T, HCG, & Beyond
Testosterone Basics & Questions
What it the purpose of Sex Hormone Binding Globulin (SHBG) ?
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<blockquote data-quote="madman" data-source="post: 218367" data-attributes="member: 13851"><p><h3>A Reappraisal of Testosterone’s Binding in Circulation: Physiological and Clinical Implications (2017)</h3><p><a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank"><em>Anna L Goldman</em></a><em>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Shalender Bhasin</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Frederick C W Wu</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Meenakshi Krishna</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Alvin M Matsumoto</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Ravi Jasuja</a></em></p><p></p><p></p><p><strong>Essential Points</strong></p><ul> <li data-xf-list-type="ul"><em><em>Most circulating testosterone is bound to its cognate binding proteins—sex hormone−binding globulin (SHBG), human serum albumin (HSA), cortisol-binding globulin, and orosomucoid; these</em><strong><em> binding proteins play an important role in regulating the transport, tissue delivery, bioactivity, and metabolism of testosterone</em></strong></em></li> </ul><p></p><p></p><p><em>Binding proteins in the peripheral circulation are important in regulating the transport, bioavailability, and metabolism of their cognate ligands, such as steroid hormones, fatty acids, vitamins, and drugs<strong>. The major sex steroid hormones—testosterone, 5α-dihydrotestosterone, and 17β-estradiol—bind predominantly to sex hormone−binding globulin (SHBG) and to human serum albumin (HSA) and to a lesser extent to corticosteroid-binding globulin (CBG) and orosomucoid. <u>SHBG, which is secreted by the liver, binds to testosterone with high affinity and is an important determinant of the distribution of circulating testosterone into its bound and free fractions</u> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">1</a>).</strong> <strong><u>HSA is one of the most abundant and versatile proteins in circulation; although it binds testosterone with lower affinity than SHBG does, its high binding capacity and high concentration allow it to buffer fluctuations in testosterone levels</u> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">1</a>). </strong>The characteristics of testosterone binding to CBG and orosomucoid and the biological roles of these binding proteins in regulating testosterone bioavailability remain incompletely understood.</em></p><p><em></em></p><p><em>Total testosterone refers to the sum of the concentrations of protein-bound and unbound testosterone in circulation. The fraction of circulating testosterone that is unbound to any plasma protein is referred to as the free testosterone fraction. The term bioavailable testosterone refers to the fraction of circulating testosterone that is not bound to SHBG and largely represents the sum of free testosterone plus HSA-bound testosterone (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Fig. 1</a>) (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">2</a>); the term reflects the view that HSA-bound testosterone, which is bound with low affinity, can dissociate from HSA in the tissue capillaries and effectively be available for biological activity. The free testosterone fraction can be measured directly by the equilibrium dialysis or ultrafiltration method or calculated from total testosterone, SHBG, and HSA concentrations using published mass action binding algorithms (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">3–6</a>). The bioavailable fraction can be measured using the ammonium sulfate precipitation method or the concanavalin A method, or it can be calculated from total testosterone, SHBG, and HSA concentrations (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">7</a>). Although the pioneers who originated the concept of bioavailable testosterone envisioned it as the sum of HSA-bound and unbound fractions of circulating testosterone (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">2</a>), the methods used to measure bioavailable testosterone concentrations, namely, the ammonium sulfate precipitation and concanavalin A methods, quantitate it as the non−SHBG-bound fraction of circulating testosterone, which approximates but is not equivalent to its original conceptualization as the sum of HSA-bound plus unbound testosterone levels (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">8</a>).</em></p><p></p><p></p><p><strong>Figure 1. <u>Partitioning of testosterone in the systemic circulation</u>. Circulating testosterone is bound tightly to SHBG (green = high-affinity binding) and weakly to albumin, orosomucoid (ORM), and CBG (blue = low-affinity binding) (11). Only 1% to 4% of circulating testosterone is unbound or free. The combination of free and albumin-bound testosterone is also referred to as the “bioavailable testosterone” fraction.</strong></p><p>[ATTACH=full]19940[/ATTACH]</p><p></p><p></p><p></p><p></p><p><strong>Biology of Binding Proteins and Their Role in the Transport, Distribution, Metabolism, and Bioavailability of Testosterone</strong></p><p><strong></strong></p><p><strong><em>At least four structurally distinct binding proteins are known to bind testosterone in human circulation: SHBG, HSA, CBG, and orosomucoid. <u>Among these, SHBG has received the most attention because of its high binding affinity for testosterone</u>.<u> These binding proteins influence the tissue bioavailability and metabolic clearance rate of testosterone by regulating the amount of free testosterone available for biological action in the tissue</u>.</em></strong><em> The roles of HSA, CBG, and orosomucoid in regulating testosterone’s bioavailability are less well understood, and we do not know how disease states or conditions that may differentially alter the circulating concentrations of HSA, CBG, and orosomucoid impact the binding of testosterone to SHBG. </em><strong><em><u>Current computations of free and bioavailable testosterone account only for the potential impact of alterations in HSA and SHBG, ignoring CBG and orosomucoid and other potentially interacting proteins and steroid hormones</u>.</em></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong></strong></p><p><strong>SHBG</strong></p><p></p><p><em>SHBG, a homodimeric glycoprotein with a molecular mass of approximately 90 kDa (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">13</a>), was first identified by Mercier et al. (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">14</a>), who separated a testosterone-binding β-globulin by electrophoresis. An estradiol-binding protein was independently isolated the same year (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">15</a>), and competitive steroid-binding studies showed that the two proteins were identical (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">16</a>). <strong>Consequently, it became known as the <u>testosterone-estradiol binding globulin</u>. This binding protein has since been shown to bind to and act as a transport protein for other sex steroid hormones as well and is, therefore, more commonly known as the SHBG (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">4</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">11</a>).</strong></em></p><p><em></em></p><p><em>The SHBG protein is encoded by a single gene on the short arm of chromosome 17, which includes eight exons (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">17</a>). Three distinct promoters—PL, PT, and PN—can initiate transcription from three separate sites in exon 1, resulting in three variants: 1L, 1T, and 1N. The typical wild-type SHBG protein is the product of translation of a transcript produced under the influence of promoter PL and the other seven exons. A variant, SHBG-T, is missing exon 7 but includes the product of exon 1T produced under the influence of promoter PT (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">18</a>).</em></p><p><em></em></p><p><em><strong>SHBG circulates as a homodimer.</strong> Calcium and zinc ions are required for holding the dimer together (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">19</a>); thus, chelating agents, such as EDTA, can dissociate the SHBG dimer.<strong> Each SHBG monomer contains two laminin G−like (LG) domains at the N-terminal end of the protein, encoded by exons 2 to 5 (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">20</a>). <u>These LG domains form pockets that enable the binding of sex hormones</u></strong>. The serine residue within this binding pocket is important in androgen and estrogen binding and forms hydrogen bonds with functional groups at the C3 position of the A ring of testosterone (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">21</a>) and with the C17 hydroxyl group in the D ring of estradiol (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">22</a>). <strong>Thus, the binding of androgens and estrogens imparts different conformations to the SHBG molecule.</strong> The SHBG protein contains three oligosaccharides; two oligosaccharides are attached at two N-glycosylation sites on asparagine and one at an O-glycosylation site on threonine (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">23</a>). <strong>SHBG levels, which typically range from 10 to 56 nmol/L, can be measured using immunofluorometric and chemiluminescent assays or by dihydrotestosterone binding assays (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">24</a>).</strong></em></p><p><em></em></p><p><em>Although reports indicate that SHBG has been produced locally in the testes, uterus, and brain, <strong>most circulating SHBG in humans is produced in the liver. </strong>The product of the SHBG gene in the testes is called the androgen-binding protein, which has different oligosaccharides and is not secreted into the circulation. SHBG production in the liver is inhibited by hepatic lipids and by tumor necrosis factor-α and interleukin-1, rather than by insulin directly, which was reported previously (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">25</a>). Thus, the low SHBG levels seen in obesity and diabetes are most likely the result of low-grade inflammation and increased amounts of hepatic lipids rather than high insulin levels (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">26</a>). Selva and Hammond have shown that thyroid hormones increase SHBG production indirectly by increasing hepatocyte nuclear 4 alpha gene expression, which is a major regulator of SHBG transcription (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">27</a>).</em></p><p><em></em></p><p><em><strong>The distribution of SHBG-bound testosterone differs in men and women: In the presence of estradiol, about 20% of binding sites are occupied by testosterone (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">11</a>).</strong> The reported association constant for binding of testosterone to SHBG has varied among published studies depending on the experimental conditions, but it is consistently reported to be around 1 × 109 L/mol with <strong>two binding sites on each SHBG homodimer (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">4</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">5</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">28–31</a>).</strong> Known variants, including the rs6258, rs143521188, rs143269613, rs146779355, and rs373769356 polymorphisms, decrease affinity for testosterone and higher equilibrium dissociation constant (Kd) values (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">32</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">33</a>). <strong>Notably, previous binding studies have assumed that the two binding sites on the SHBG homodimer are equivalent. <u>A recent reappraisal of testosterone binding to SHBG using modern biophysical techniques indicated that the two binding sites on the SHBG dimer are not equivalent and that there is an allosteric interaction between the binding sites on the SHBG dimer such that the second testosterone molecule binds SHBG with a substantially different affinity than the first binding site</u> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">34</a>). The allosteric model of the multistep binding of testosterone to SHBG is discussed later in this review.</strong></em></p><p></p><p></p><p></p><p></p><p><strong>Additional Potential Roles of SHBG and Orosomucoid</strong></p><p></p><p><em><strong>The classic genomic signaling that mediates the biological actions of testosterone involves its passive diffusion into the cellular cytoplasm [<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Fig. 4(a</a>)], association with the androgen receptor, translocation into the nucleus, and binding to the DNA response element to modulate transcription of specific androgen-responsive genes. Although passive diffusion is widely observed in multiple cell types, the globulin family proteins are postulated to facilitate cellular steroid uptake [<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Fig. 4(b)–4(d</a>)]. <u>Binding proteins, such as SHBG, have been described as multifunctional proteins, capable of regulating the response to steroid hormones as well as their entry into cells</u> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">13</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">77–79</a>). <u>These binding proteins are also postulated to serve other functions, as described later</u> (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">80–88</a>).</strong></em></p><p></p><p></p><p><strong>Figure 4. Multiple hypothetical mechanisms for the cellular uptake of testosterone and downstream signaling. (a) <u>The model depicts the “free” hormone hypothesis</u>. </strong>In this model, testosterone (T) that is not bound to SHBG or HSA or other binding proteins diffuses across the plasma membrane and binds to the androgen receptor (AR). The liganded AR recruits coregulators and chaperone proteins translocate to the nucleus and bind to androgen response elements (AREs) on androgen-responsive target genes, which activates the transcription of target genes.<strong> (b) <u>The megalin-dependent mode of testosterone entry</u>. </strong>According to this model, SHBG-bound testosterone is internalized into the cell through an endocytic process mediated by the membrane protein megalin. Once internalized, SHBG-bound testosterone is released at the low pH within the lysosome.<strong> (c) <u>The SHBG receptor-testosterone system</u>. </strong>The SHBG dimer has multiple binding sites—two sites (simplified as one in this model) bind testosterone, and one site binds to a membrane receptor. It may be that only unbound SHBG is able to bind to the receptor, then the SHBG-receptor-testosterone complex is coupled to the activation of a G protein (GP), the accumulation of intracellular cyclic adenosine monophosphate (cAMP), and activation of protein kinase A (PKA). PKA may modulate AR function by activating AR through phosphorylation (not depicted) (92).<strong> (d) <u>Steroid ligand2dependent interactions between SHBG and at least two matrix-associated proteins in the fibulin family (fibulin-1D and fibulin-2)</u> </strong>contribute to the extravascular sequestration of SHBG in some tissues, such as the breast, prostate, and endometrial stroma. According to this model, ligand-dependent interactions between SHBG and fibulins modulate their binding to various signaling molecules, such as integrins, to modify signaling pathways that regulate cell adhesion, proliferation, and migration. mRNA, messenger RNA.</p><p>[ATTACH=full]19941[/ATTACH]</p><p>[ATTACH=full]19942[/ATTACH]</p><p></p><p></p><p></p><p></p><p><strong>Potential role of SHBG in the prostate</strong></p><p></p><p><em><strong>In the 1990s, several investigators reported that SHBG might bind to cell surface binding sites on prostate cells and activate intracellular signaling on its own [<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Fig. 4(c</a>)] (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">89–91</a>).</strong> However, the cell surface receptors for SHBG have not been isolated or fully characterized. Therefore, we do not know whether SHBG has an independent role in regulating prostate growth or function. <strong>The postulated SHBG receptor−testosterone system, as well as the megalin-dependent transport of testosterone into the cell, are discussed in later sections [<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Fig. 4(b</a>)].</strong></em></p><p></p><p></p><p></p><p></p><p><strong>Potential role of SHBG and fibulins in the endometrium</strong></p><p></p><p><em>Fibulins are secreted glycoproteins in the blood and extracellular matrix that act as bridging peptides between elastin fibers and cell surface integrins and become incorporated into the fibrillar extracellular matrix. There are seven members of the fibulin family, each with a different biological role. <strong>Steroid hormone−dependent interactions between SHBG and at least two fibulin family members (fibulin-1D and fibulin-2) may contribute to the extravascular accumulation and distribution of SHBG within the endometrial stroma, where it has been reported to control sex steroid access to target cells (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">93</a>).</strong> <strong>This interaction may provide a molecular scaffold for signaling molecules such as integrins and represents a new mechanism of steroid hormone action [<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Fig. 4(d</a>)] (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">93–96</a>).</strong> These protein−protein interactions suggest additional regulation of the bioavailability of testosterone at the tissue level through tissue-binding proteins such as fibulins.</em></p><p></p><p></p><p></p><p></p><p><strong>Circulating SHBG level as a biomarker of metabolic risk</strong></p><p></p><p><em>In epidemiologic studies, low total testosterone levels have been associated with increased risks of diabetes and metabolic syndrome, a cluster of conditions including hypertension, insulin resistance, central obesity, and dyslipidemia, which predispose individuals to an increased risk of cardiovascular disease. In longitudinal analyses, SHBG levels rather than total or free testosterone levels have been independently and prospectively associated with incident diabetes and metabolic syndrome after adjustments for age, adiposity, and comorbid conditions (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">97</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">98</a>). Among children and adolescents, SHBG may also be a biomarker for metabolic syndrome risk (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">99</a>), and lower levels were more robustly associated with the risk of metabolic syndrome in boys than in girls (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">100</a>). <strong>We do not know whether SHBG is merely a marker of metabolic risk or whether SHBG plays a causal role in the pathophysiology of metabolic disorders such as diabetes and metabolic syndrome.</strong></em></p><p></p><p></p><p></p><p></p><p><strong>Role of orosomucoid in acute and chronic infections</strong></p><p></p><p><em>Orosomucoid, an acute phase reactant, evolved from the immunoglobulin protein superfamily (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">101</a>). Inflammatory modulators, such as cytokines and chemokines, influence the expression of the AGP gene and orosomucoid synthesis (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">102</a>). Circulating orosomucoid concentrations are increased in the setting of infection (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">103</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">104</a>), and orosomucoid was recently established as an effective prognostic marker of the severity of sepsis (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">105</a>). Orosomucoid plays an important role in the inflammatory response by inhibiting neutrophil migration in sepsis through a nitric oxide−dependent mechanism (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">103</a>). It may also have a protective function by binding to lipopolysaccharide and enhancing its clearance from the body (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">106</a>) and by inhibiting platelet aggregation to prevent hypercoagulability in sepsis (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">107</a>, <a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">108</a>). Orosomucoid has also been reported to regulate the bioavailability of protease inhibitors in persons with chronic HIV infection (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">109</a>), which may have important implications for therapeutic drug monitoring (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">110</a>). <strong>Orosomucoid may play a similar role in the distribution and bioavailability of testosterone in persons infected with HIV or hepatitis C virus (HCV), who often display marked alterations in binding protein (<a href="https://www.excelmale.com/forum/javascript%3A;" target="_blank">Table 2</a>) concentrations.</strong></em></p><p></p><p></p><p></p><p></p><h2>Synthesis</h2><p><em><strong>Sex steroid bioactivity and the respective roles of SHBG and HSA are more complex than originally believed. </strong>The oversimplified assumptions of stoichiometry, binding dynamics, and binding affinity have contributed to the development of inaccurate linear binding models, which have been propagated without much critical reappraisal until now. These historical linear models and the resulting equations for calculating free testosterone based on these legacy models are widely used and may potentially increase the risk of misclassifying men seeking testosterone therapy. A novel multistep EAM of the binding of testosterone to SHBG provides a close approximation of free testosterone levels using equilibrium dialysis, but clinical experience with this new model is currently limited. Harmonized reference ranges for free testosterone are needed to demarcate individuals who are eugonadal from those who are hypogonadal, acknowledging that different symptoms may have different thresholds. These steps would reduce the risk of disease misclassification and optimize clinical decision-making in the management of androgen disorders in men and women.</em></p></blockquote><p></p>
[QUOTE="madman, post: 218367, member: 13851"] [HEADING=2]A Reappraisal of Testosterone’s Binding in Circulation: Physiological and Clinical Implications (2017)[/HEADING] [URL='https://www.excelmale.com/forum/javascript%3A;'][I]Anna L Goldman[/I][/URL][I], [URL='https://www.excelmale.com/forum/javascript%3A;']Shalender Bhasin[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']Frederick C W Wu[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']Meenakshi Krishna[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']Alvin M Matsumoto[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']Ravi Jasuja[/URL][/I] [B]Essential Points[/B] [LIST] [*][I][I]Most circulating testosterone is bound to its cognate binding proteins—sex hormone−binding globulin (SHBG), human serum albumin (HSA), cortisol-binding globulin, and orosomucoid; these[/I][B][I] binding proteins play an important role in regulating the transport, tissue delivery, bioactivity, and metabolism of testosterone[/I][/B][/I] [/LIST] [I]Binding proteins in the peripheral circulation are important in regulating the transport, bioavailability, and metabolism of their cognate ligands, such as steroid hormones, fatty acids, vitamins, and drugs[B]. The major sex steroid hormones—testosterone, 5α-dihydrotestosterone, and 17β-estradiol—bind predominantly to sex hormone−binding globulin (SHBG) and to human serum albumin (HSA) and to a lesser extent to corticosteroid-binding globulin (CBG) and orosomucoid. [U]SHBG, which is secreted by the liver, binds to testosterone with high affinity and is an important determinant of the distribution of circulating testosterone into its bound and free fractions[/U] ([URL='https://www.excelmale.com/forum/javascript%3A;']1[/URL]).[/B] [B][U]HSA is one of the most abundant and versatile proteins in circulation; although it binds testosterone with lower affinity than SHBG does, its high binding capacity and high concentration allow it to buffer fluctuations in testosterone levels[/U] ([URL='https://www.excelmale.com/forum/javascript%3A;']1[/URL]). [/B]The characteristics of testosterone binding to CBG and orosomucoid and the biological roles of these binding proteins in regulating testosterone bioavailability remain incompletely understood. Total testosterone refers to the sum of the concentrations of protein-bound and unbound testosterone in circulation. The fraction of circulating testosterone that is unbound to any plasma protein is referred to as the free testosterone fraction. The term bioavailable testosterone refers to the fraction of circulating testosterone that is not bound to SHBG and largely represents the sum of free testosterone plus HSA-bound testosterone ([URL='https://www.excelmale.com/forum/javascript%3A;']Fig. 1[/URL]) ([URL='https://www.excelmale.com/forum/javascript%3A;']2[/URL]); the term reflects the view that HSA-bound testosterone, which is bound with low affinity, can dissociate from HSA in the tissue capillaries and effectively be available for biological activity. The free testosterone fraction can be measured directly by the equilibrium dialysis or ultrafiltration method or calculated from total testosterone, SHBG, and HSA concentrations using published mass action binding algorithms ([URL='https://www.excelmale.com/forum/javascript%3A;']3–6[/URL]). The bioavailable fraction can be measured using the ammonium sulfate precipitation method or the concanavalin A method, or it can be calculated from total testosterone, SHBG, and HSA concentrations ([URL='https://www.excelmale.com/forum/javascript%3A;']7[/URL]). Although the pioneers who originated the concept of bioavailable testosterone envisioned it as the sum of HSA-bound and unbound fractions of circulating testosterone ([URL='https://www.excelmale.com/forum/javascript%3A;']2[/URL]), the methods used to measure bioavailable testosterone concentrations, namely, the ammonium sulfate precipitation and concanavalin A methods, quantitate it as the non−SHBG-bound fraction of circulating testosterone, which approximates but is not equivalent to its original conceptualization as the sum of HSA-bound plus unbound testosterone levels ([URL='https://www.excelmale.com/forum/javascript%3A;']8[/URL]).[/I] [B]Figure 1. [U]Partitioning of testosterone in the systemic circulation[/U]. Circulating testosterone is bound tightly to SHBG (green = high-affinity binding) and weakly to albumin, orosomucoid (ORM), and CBG (blue = low-affinity binding) (11). Only 1% to 4% of circulating testosterone is unbound or free. The combination of free and albumin-bound testosterone is also referred to as the “bioavailable testosterone” fraction.[/B] [ATTACH type="full" alt="1645841158590.png"]19940[/ATTACH] [B]Biology of Binding Proteins and Their Role in the Transport, Distribution, Metabolism, and Bioavailability of Testosterone [I]At least four structurally distinct binding proteins are known to bind testosterone in human circulation: SHBG, HSA, CBG, and orosomucoid. [U]Among these, SHBG has received the most attention because of its high binding affinity for testosterone[/U].[U] These binding proteins influence the tissue bioavailability and metabolic clearance rate of testosterone by regulating the amount of free testosterone available for biological action in the tissue[/U].[/I][/B][I] The roles of HSA, CBG, and orosomucoid in regulating testosterone’s bioavailability are less well understood, and we do not know how disease states or conditions that may differentially alter the circulating concentrations of HSA, CBG, and orosomucoid impact the binding of testosterone to SHBG. [/I][B][I][U]Current computations of free and bioavailable testosterone account only for the potential impact of alterations in HSA and SHBG, ignoring CBG and orosomucoid and other potentially interacting proteins and steroid hormones[/U].[/I] SHBG[/B] [I]SHBG, a homodimeric glycoprotein with a molecular mass of approximately 90 kDa ([URL='https://www.excelmale.com/forum/javascript%3A;']13[/URL]), was first identified by Mercier et al. ([URL='https://www.excelmale.com/forum/javascript%3A;']14[/URL]), who separated a testosterone-binding β-globulin by electrophoresis. An estradiol-binding protein was independently isolated the same year ([URL='https://www.excelmale.com/forum/javascript%3A;']15[/URL]), and competitive steroid-binding studies showed that the two proteins were identical ([URL='https://www.excelmale.com/forum/javascript%3A;']16[/URL]). [B]Consequently, it became known as the [U]testosterone-estradiol binding globulin[/U]. This binding protein has since been shown to bind to and act as a transport protein for other sex steroid hormones as well and is, therefore, more commonly known as the SHBG ([URL='https://www.excelmale.com/forum/javascript%3A;']4[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']11[/URL]).[/B] The SHBG protein is encoded by a single gene on the short arm of chromosome 17, which includes eight exons ([URL='https://www.excelmale.com/forum/javascript%3A;']17[/URL]). Three distinct promoters—PL, PT, and PN—can initiate transcription from three separate sites in exon 1, resulting in three variants: 1L, 1T, and 1N. The typical wild-type SHBG protein is the product of translation of a transcript produced under the influence of promoter PL and the other seven exons. A variant, SHBG-T, is missing exon 7 but includes the product of exon 1T produced under the influence of promoter PT ([URL='https://www.excelmale.com/forum/javascript%3A;']18[/URL]). [B]SHBG circulates as a homodimer.[/B] Calcium and zinc ions are required for holding the dimer together ([URL='https://www.excelmale.com/forum/javascript%3A;']19[/URL]); thus, chelating agents, such as EDTA, can dissociate the SHBG dimer.[B] Each SHBG monomer contains two laminin G−like (LG) domains at the N-terminal end of the protein, encoded by exons 2 to 5 ([URL='https://www.excelmale.com/forum/javascript%3A;']20[/URL]). [U]These LG domains form pockets that enable the binding of sex hormones[/U][/B]. The serine residue within this binding pocket is important in androgen and estrogen binding and forms hydrogen bonds with functional groups at the C3 position of the A ring of testosterone ([URL='https://www.excelmale.com/forum/javascript%3A;']21[/URL]) and with the C17 hydroxyl group in the D ring of estradiol ([URL='https://www.excelmale.com/forum/javascript%3A;']22[/URL]). [B]Thus, the binding of androgens and estrogens imparts different conformations to the SHBG molecule.[/B] The SHBG protein contains three oligosaccharides; two oligosaccharides are attached at two N-glycosylation sites on asparagine and one at an O-glycosylation site on threonine ([URL='https://www.excelmale.com/forum/javascript%3A;']23[/URL]). [B]SHBG levels, which typically range from 10 to 56 nmol/L, can be measured using immunofluorometric and chemiluminescent assays or by dihydrotestosterone binding assays ([URL='https://www.excelmale.com/forum/javascript%3A;']24[/URL]).[/B] Although reports indicate that SHBG has been produced locally in the testes, uterus, and brain, [B]most circulating SHBG in humans is produced in the liver. [/B]The product of the SHBG gene in the testes is called the androgen-binding protein, which has different oligosaccharides and is not secreted into the circulation. SHBG production in the liver is inhibited by hepatic lipids and by tumor necrosis factor-α and interleukin-1, rather than by insulin directly, which was reported previously ([URL='https://www.excelmale.com/forum/javascript%3A;']25[/URL]). Thus, the low SHBG levels seen in obesity and diabetes are most likely the result of low-grade inflammation and increased amounts of hepatic lipids rather than high insulin levels ([URL='https://www.excelmale.com/forum/javascript%3A;']26[/URL]). Selva and Hammond have shown that thyroid hormones increase SHBG production indirectly by increasing hepatocyte nuclear 4 alpha gene expression, which is a major regulator of SHBG transcription ([URL='https://www.excelmale.com/forum/javascript%3A;']27[/URL]). [B]The distribution of SHBG-bound testosterone differs in men and women: In the presence of estradiol, about 20% of binding sites are occupied by testosterone ([URL='https://www.excelmale.com/forum/javascript%3A;']11[/URL]).[/B] The reported association constant for binding of testosterone to SHBG has varied among published studies depending on the experimental conditions, but it is consistently reported to be around 1 × 109 L/mol with [B]two binding sites on each SHBG homodimer ([URL='https://www.excelmale.com/forum/javascript%3A;']4[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']5[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']28–31[/URL]).[/B] Known variants, including the rs6258, rs143521188, rs143269613, rs146779355, and rs373769356 polymorphisms, decrease affinity for testosterone and higher equilibrium dissociation constant (Kd) values ([URL='https://www.excelmale.com/forum/javascript%3A;']32[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']33[/URL]). [B]Notably, previous binding studies have assumed that the two binding sites on the SHBG homodimer are equivalent. [U]A recent reappraisal of testosterone binding to SHBG using modern biophysical techniques indicated that the two binding sites on the SHBG dimer are not equivalent and that there is an allosteric interaction between the binding sites on the SHBG dimer such that the second testosterone molecule binds SHBG with a substantially different affinity than the first binding site[/U] ([URL='https://www.excelmale.com/forum/javascript%3A;']34[/URL]). The allosteric model of the multistep binding of testosterone to SHBG is discussed later in this review.[/B][/I] [B]Additional Potential Roles of SHBG and Orosomucoid[/B] [I][B]The classic genomic signaling that mediates the biological actions of testosterone involves its passive diffusion into the cellular cytoplasm [[URL='https://www.excelmale.com/forum/javascript%3A;']Fig. 4(a[/URL])], association with the androgen receptor, translocation into the nucleus, and binding to the DNA response element to modulate transcription of specific androgen-responsive genes. Although passive diffusion is widely observed in multiple cell types, the globulin family proteins are postulated to facilitate cellular steroid uptake [[URL='https://www.excelmale.com/forum/javascript%3A;']Fig. 4(b)–4(d[/URL])]. [U]Binding proteins, such as SHBG, have been described as multifunctional proteins, capable of regulating the response to steroid hormones as well as their entry into cells[/U] ([URL='https://www.excelmale.com/forum/javascript%3A;']13[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']77–79[/URL]). [U]These binding proteins are also postulated to serve other functions, as described later[/U] ([URL='https://www.excelmale.com/forum/javascript%3A;']80–88[/URL]).[/B][/I] [B]Figure 4. Multiple hypothetical mechanisms for the cellular uptake of testosterone and downstream signaling. (a) [U]The model depicts the “free” hormone hypothesis[/U]. [/B]In this model, testosterone (T) that is not bound to SHBG or HSA or other binding proteins diffuses across the plasma membrane and binds to the androgen receptor (AR). The liganded AR recruits coregulators and chaperone proteins translocate to the nucleus and bind to androgen response elements (AREs) on androgen-responsive target genes, which activates the transcription of target genes.[B] (b) [U]The megalin-dependent mode of testosterone entry[/U]. [/B]According to this model, SHBG-bound testosterone is internalized into the cell through an endocytic process mediated by the membrane protein megalin. Once internalized, SHBG-bound testosterone is released at the low pH within the lysosome.[B] (c) [U]The SHBG receptor-testosterone system[/U]. [/B]The SHBG dimer has multiple binding sites—two sites (simplified as one in this model) bind testosterone, and one site binds to a membrane receptor. It may be that only unbound SHBG is able to bind to the receptor, then the SHBG-receptor-testosterone complex is coupled to the activation of a G protein (GP), the accumulation of intracellular cyclic adenosine monophosphate (cAMP), and activation of protein kinase A (PKA). PKA may modulate AR function by activating AR through phosphorylation (not depicted) (92).[B] (d) [U]Steroid ligand2dependent interactions between SHBG and at least two matrix-associated proteins in the fibulin family (fibulin-1D and fibulin-2)[/U] [/B]contribute to the extravascular sequestration of SHBG in some tissues, such as the breast, prostate, and endometrial stroma. According to this model, ligand-dependent interactions between SHBG and fibulins modulate their binding to various signaling molecules, such as integrins, to modify signaling pathways that regulate cell adhesion, proliferation, and migration. mRNA, messenger RNA. [ATTACH type="full" alt="Screenshot (11133).png"]19941[/ATTACH] [ATTACH type="full" alt="Screenshot (11134).png"]19942[/ATTACH] [B]Potential role of SHBG in the prostate[/B] [I][B]In the 1990s, several investigators reported that SHBG might bind to cell surface binding sites on prostate cells and activate intracellular signaling on its own [[URL='https://www.excelmale.com/forum/javascript%3A;']Fig. 4(c[/URL])] ([URL='https://www.excelmale.com/forum/javascript%3A;']89–91[/URL]).[/B] However, the cell surface receptors for SHBG have not been isolated or fully characterized. Therefore, we do not know whether SHBG has an independent role in regulating prostate growth or function. [B]The postulated SHBG receptor−testosterone system, as well as the megalin-dependent transport of testosterone into the cell, are discussed in later sections [[URL='https://www.excelmale.com/forum/javascript%3A;']Fig. 4(b[/URL])].[/B][/I] [B]Potential role of SHBG and fibulins in the endometrium[/B] [I]Fibulins are secreted glycoproteins in the blood and extracellular matrix that act as bridging peptides between elastin fibers and cell surface integrins and become incorporated into the fibrillar extracellular matrix. There are seven members of the fibulin family, each with a different biological role. [B]Steroid hormone−dependent interactions between SHBG and at least two fibulin family members (fibulin-1D and fibulin-2) may contribute to the extravascular accumulation and distribution of SHBG within the endometrial stroma, where it has been reported to control sex steroid access to target cells ([URL='https://www.excelmale.com/forum/javascript%3A;']93[/URL]).[/B] [B]This interaction may provide a molecular scaffold for signaling molecules such as integrins and represents a new mechanism of steroid hormone action [[URL='https://www.excelmale.com/forum/javascript%3A;']Fig. 4(d[/URL])] ([URL='https://www.excelmale.com/forum/javascript%3A;']93–96[/URL]).[/B] These protein−protein interactions suggest additional regulation of the bioavailability of testosterone at the tissue level through tissue-binding proteins such as fibulins.[/I] [B]Circulating SHBG level as a biomarker of metabolic risk[/B] [I]In epidemiologic studies, low total testosterone levels have been associated with increased risks of diabetes and metabolic syndrome, a cluster of conditions including hypertension, insulin resistance, central obesity, and dyslipidemia, which predispose individuals to an increased risk of cardiovascular disease. In longitudinal analyses, SHBG levels rather than total or free testosterone levels have been independently and prospectively associated with incident diabetes and metabolic syndrome after adjustments for age, adiposity, and comorbid conditions ([URL='https://www.excelmale.com/forum/javascript%3A;']97[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']98[/URL]). Among children and adolescents, SHBG may also be a biomarker for metabolic syndrome risk ([URL='https://www.excelmale.com/forum/javascript%3A;']99[/URL]), and lower levels were more robustly associated with the risk of metabolic syndrome in boys than in girls ([URL='https://www.excelmale.com/forum/javascript%3A;']100[/URL]). [B]We do not know whether SHBG is merely a marker of metabolic risk or whether SHBG plays a causal role in the pathophysiology of metabolic disorders such as diabetes and metabolic syndrome.[/B][/I] [B]Role of orosomucoid in acute and chronic infections[/B] [I]Orosomucoid, an acute phase reactant, evolved from the immunoglobulin protein superfamily ([URL='https://www.excelmale.com/forum/javascript%3A;']101[/URL]). Inflammatory modulators, such as cytokines and chemokines, influence the expression of the AGP gene and orosomucoid synthesis ([URL='https://www.excelmale.com/forum/javascript%3A;']102[/URL]). Circulating orosomucoid concentrations are increased in the setting of infection ([URL='https://www.excelmale.com/forum/javascript%3A;']103[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']104[/URL]), and orosomucoid was recently established as an effective prognostic marker of the severity of sepsis ([URL='https://www.excelmale.com/forum/javascript%3A;']105[/URL]). Orosomucoid plays an important role in the inflammatory response by inhibiting neutrophil migration in sepsis through a nitric oxide−dependent mechanism ([URL='https://www.excelmale.com/forum/javascript%3A;']103[/URL]). It may also have a protective function by binding to lipopolysaccharide and enhancing its clearance from the body ([URL='https://www.excelmale.com/forum/javascript%3A;']106[/URL]) and by inhibiting platelet aggregation to prevent hypercoagulability in sepsis ([URL='https://www.excelmale.com/forum/javascript%3A;']107[/URL], [URL='https://www.excelmale.com/forum/javascript%3A;']108[/URL]). Orosomucoid has also been reported to regulate the bioavailability of protease inhibitors in persons with chronic HIV infection ([URL='https://www.excelmale.com/forum/javascript%3A;']109[/URL]), which may have important implications for therapeutic drug monitoring ([URL='https://www.excelmale.com/forum/javascript%3A;']110[/URL]). [B]Orosomucoid may play a similar role in the distribution and bioavailability of testosterone in persons infected with HIV or hepatitis C virus (HCV), who often display marked alterations in binding protein ([URL='https://www.excelmale.com/forum/javascript%3A;']Table 2[/URL]) concentrations.[/B][/I] [HEADING=1]Synthesis[/HEADING] [I][B]Sex steroid bioactivity and the respective roles of SHBG and HSA are more complex than originally believed. [/B]The oversimplified assumptions of stoichiometry, binding dynamics, and binding affinity have contributed to the development of inaccurate linear binding models, which have been propagated without much critical reappraisal until now. These historical linear models and the resulting equations for calculating free testosterone based on these legacy models are widely used and may potentially increase the risk of misclassifying men seeking testosterone therapy. A novel multistep EAM of the binding of testosterone to SHBG provides a close approximation of free testosterone levels using equilibrium dialysis, but clinical experience with this new model is currently limited. Harmonized reference ranges for free testosterone are needed to demarcate individuals who are eugonadal from those who are hypogonadal, acknowledging that different symptoms may have different thresholds. These steps would reduce the risk of disease misclassification and optimize clinical decision-making in the management of androgen disorders in men and women.[/I] [/QUOTE]
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Testosterone Replacement, Low T, HCG, & Beyond
Testosterone Basics & Questions
What it the purpose of Sex Hormone Binding Globulin (SHBG) ?
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