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Biomedical subjects

W Rosner

Publications and source records attributed to W Rosner.

At least 37 records · Page 2Linked to original sources
PubMed1994

The relationship between serum levels of insulin and sex hormone-binding globulin in men: the effect of weight loss.

It is known that there is an inverse relationship between the serum levels of insulin and sex hormone-binding globulin (SHBG) in women, but the relationship in men has not been reported. It is not known whether changes in the one cause changes in the other, or whether they change in opposite directions in response to some third factor. Because obesity raises insulin levels and lowers SHBG levels in both sexes, we proposed to study the cause-effect question by determining whether the relationship between changes in SHBG and insulin levels during active weight loss. We studied 70 healthy weight-stable men with body mass index (BMI) from 20.7-94 (normal, 22.5 +/- 2.5) and restudied 17 of them during diet-induced weight loss. Fasting serum insulin levels in the weight-stable men showed a positive linear correlation with BMI, increasing 1 microU/mL per unit increase in BMI (P < 0.0001). SHBG levels in the weight-stable men showed a negative linear correlation with BMI, decreasing 0.2 nmol/L per unit increase in BMI (P < 0.0002). In the weight-stable men, there was an inverse hyperbolic correlation between SHBG and insulin levels; SHBG (nmol/L) = 13.1 + [30.1 divided by insulin (microU/mL)] (P < 0.002). During weight loss, insulin levels decreased at an average rate of 6.1 microU/mL per unit decrease in BMI, a much higher slope than the positive slope vs. BMI in weight stable men. During weight loss, SHBG levels increased at an average slope of 0.43 nmol/L per unit decrease in BMI, much higher than the negative slope of 0.2 nmol/L per unit increase in BMI in weight-stable men. Values for the SHBG vs. insulin coordinates in the weight-losing subjects did not differ significantly from those expected from the SHBG vs. insulin equation in weight-stable subjects. The stability of the SHBG-insulin relationship during weight loss despite the profoundly altered relationship of each separate component to BMI strongly suggests a close metabolic link between SHBG and insulin. As SHBG is not known to alter the production or metabolism of insulin, whereas insulin has been shown in vitro to decrease the synthesis of SHBG, it seems a reasonable conclusion that the predictable inverse relationship between serum insulin and SHBG indicates that insulin controls SHBG synthesis in vivo.

Body Mass Index↗
PubMed1993

Corticosteroid-binding globulin receptor of the rat hepatic membrane: solubilization, partial characterization, and the effect of steroids on binding.

The corticosteroid-binding globulin (CBG) receptor of rat hepatic membranes was solubilized using 1.5% Triton X-100. An assay for its activity was developed that was dependent upon the fact that the [125I] CBG-receptor complex adsorbs to hydroxylapatite, whereas [125I]CBG does not. Scatchard analysis of the soluble receptor at 37 C showed a single set of high affinity binding sites, with a Kd of 44 nM and a binding capacity of 7.3 pmol/mg protein. The association rate constant (k1) was 0.92 x 10(5) M-1 min-1 at 37 C, and the dissociation rate constant (k2) was 1.0 x 10(-3) min-1. Only unliganded CBG could bind to the receptor. Steroids that bound to CBG, e.g. corticosterone and cortisol, noncompetitively inhibited CBG's binding to the receptor. Steroids that did not bind to CBG, e.g, dexamethasone, were without effect on the interaction of CBG with its receptor.

Animals↗
PubMed1992

Comparison of the carbohydrate composition of rat and human corticosteroid-binding globulin: species specific glycosylation.

We have examined the carbohydrate composition of corticosteroid-binding globulin (CBG) obtained from rat and human serum. Rat CBG contained a carbohydrate composition that was strikingly different from that of human CBG. Like other glycoproteins that circulate in human plasma, human CBG had a carbohydrate composition that was consistent with the presence of biantennary and triantennary oligosaccharide structures. In contrast, the carbohydrate composition of rat CBG indicated the presence of more than one sialic acid residue per antenna. It is not clear whether rat CBG contains a carbohydrate structure with sialic acids attached to both galactose and N-acetylglucosamine on the same antenna, or a terminal disialylated structure (sialic acid linked alpha 2-8 to sialic acid). These structural variations may play a role in the interaction of CBG with its receptor.

Animals↗
PubMed1991

Sex hormone-binding globulin: anatomy and physiology of a new regulatory system.

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that binds a number of circulating steroid hormones (testosterone, dihydrotestosterone and estradiol) with high affinity, thus regulating their free concentration in plasma. In addition to binding steroids, SHBG itself binds to receptor sites on plasma membranes with somewhat unusual kinetics. Both the off and on rates are quite slow. The steroid-binding and membrane-binding functions are intertwined in what is clearly an allosteric relationship. Occupation of SHBG's steroid-binding site by a steroid inhibits its ability to bind to its membrane receptor-binding site. This inhibition is not related to a steroid's biological activity. Metabolites of steroids without biological activity, e.g. 2-methoxyestradiol, actively inhibit SHBG's interaction with its membrane receptor. However, if unliganded SHBG is allowed to bind to its receptor on intact cells, and an appropriate steroid hormone then is introduced, adenylate cyclase is activated and intracellular cAMP increases. This function is specific for steroids with biological activity, 2-methoxyestradiol has no activity in this arena. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.

Amino Acid Sequence↗
PubMed1991

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 prostate, 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.

Biological Transport↗
PubMed1991

Plasma steroid-binding proteins.

Two steroid-binding proteins circulate in plasma, corticosteroid-binding globulin and sex hormone-binding globulin. They both have several different but connected, physiologic functions. Each is the major determinant of the concentration of the physiologically important hormones that they bind. CBG regulates the concentration of free cortisol and progesterone, and SHBG regulates the concentration of free testosterone, dihydrotestosterone, and, to a lesser extent, estradiol. It is this small free fraction of the appropriate hormone that is the active principal in affecting hormone action. In the past few years, it has been shown that both of these proteins have high affinity, specific receptors on the plasma membranes of a variety of cells. It has also been shown that when SHBG's binding sites are occupied it cannot bind to its receptor; only unliganded SHBG can. There are, as yet, no published reports on the control of CBG binding by steroids. For both SHBG and CBG, if an appropriate steroid is present when the binding protein is itself bound to its receptor, rapid induction of adenylate cyclase activity and the accumulation of intracellular cAMP occur. Finally, CBG has been shown to be a member of the superfamily of serine proteinase inhibitors. When it is exposed to a serine protease, it is cleaved and release all, or most, of its bound cortisol.

Animals↗
PubMed1990

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

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

Amino Acid Sequence↗
PubMed1990

Histidine 235 of human sex hormone-binding globulin is the covalent site of attachment of the nucleophilic steroid derivative, 17 beta-bromoacetoxydihydrotestosterone.

This article deals with the elucidation of the steroid-binding site of human sex hormone-binding globulin (SHBG). 17 beta-Bromoacetoxydihydrotesterone (BA-DHT) reacted with highly purified SHBG in a time-dependent and irreversible fashion. The interaction could be totally inhibited by the simultaneous addition of an excess of dihydrotesterone. At the completion of the reaction, the molar ratio of BA-DHT to SHBG was approximately unity. SHBG was affinity labeled with [14C]BA-DHT and submitted to acid hydrolysis. The released amino acids were evaluated on high performance liquid chromatography, and virtually all of the 14C was identified as 3-[14C]carboxymethylhistidine. Furthermore, [14C]BA-DHT-labeled SHBG was digested with trypsin, followed by isolation of the released tryptic peptides by reverse-phase high performance liquid chromatography. The 14C was localized to a single tryptic peptide. It contained 2' histidyl residues, corresponding to residues 235 and 251 in the known amino acid sequence of SHBG. Although most of the 3-[14C]carboxymethylhistidine, or its phenylthiohydantoin derivative, was trapped on the filter of the amino acid sequenator, sufficient radioactivity emerged to identify histidyl residue 235 as the labeled amino acid.

Affinity Labels↗
PubMed1990

The control of the interaction of sex hormone-binding globulin with its receptor by steroid hormones.

Sex hormone-binding globulins (SHBG) is a plasma glycoprotein that binds certain steroids. It, in turn, binds to a specific receptor on cell membranes. This work was undertaken to investigate the role of steroids in the interaction of SHBG with its receptor. Because the probe for the interaction of SHBG with its receptor is 125I-SHBG, we first showed that 125I-SHBG binds [3H]dihydrotestosterone (DHT) at 4 degrees C and 37 degrees C with KD values similar to those published previously for pure radioinert SHBG. 125I-SHBG could be prevented from binding to its receptor by a variety of steroids whose relative inhibitory activity (dihydrotestosterone much greater than 2-methoxyestradiol greater than testosterone greater than estradiol much greater than methyltrienolone greater than cortisol) was almost identical to their relative ability to bind to SHBG. Because significant binding of [3H]DHT to the SHBG receptor could not be demonstrated, steroid inhibition of SHBG binding must be noncompetitive. If steroids bound to SHBG prevent binding to the SHBG receptor, then liganded SHBG should have a higher apparent KD for its receptor than unliganded SHBG. This is the case. The KD was 0.86 +/- 0.25 nM for the high affinity receptor site using liganded SHBG and 0.19 +/- 0.024 nM for unliganded SHBG. Thus, only liganded SHBG assumes a conformation that prohibits interaction with the SHBG receptor. However, when unliganded SHBG was prebound to its receptor, it retained its ability to bind [3H] DHT. The model that emerges from these observations is as follows. Unliganded SHBG can bind either steroids or receptor in a reversible reaction; SHBG bound to a steroid cannot bind to the receptor, but unliganded SHBG that first binds to the receptor can subsequently bind steroids.

Binding Sites↗
PubMed1990

The rat hepatic corticosteroid-binding globulin receptor: distinction from the asialoglycoprotein receptor.

This investigation was undertaken to ascertain whether rat liver cells contained a receptor for corticosteroid-binding globulin (CBG) that could be differentiated clearly from the asialoglycoprotein receptor. To do this, [125I]CBG, [125I] asialo-CBG, and [125I]asialofetuin were used as probes to differentiate the binding activities of the two receptors. On hepatic membranes, CBG bound to a single set of sites with a Kd of 0.74 microM, asialofetuin bound to a single set of sites with a Kd of 0.018 microM, asialo-CBG bound to two sets of sites with Kd values of 0.004 and 1.4 microM and in the presence of 1 microM asialofetium, asialo-CBG bound to a single set of sites with a Kd of 0.53 microM, not different (P greater than 0.2) from the Kd of CBG. Cross-competition studies using the three 125I-labeled ligands and allowing each to compete with the three radioinert ligands indicated the existence of two separate receptors. Desialylation of hepatic membranes differentially affected the binding of CBG and asialofetuin. Finally, whole cells bound CBG specifically, but internalized it to only a minimal extent (less than 10%). This observation does not support a role for the CBG-receptor system in the entry of steroids into cells.

Animals↗
PubMed1990

Biologically active steroids activate receptor-bound human sex hormone-binding globulin to cause LNCaP cells to accumulate adenosine 3',5'-monophosphate.

The binding of human sex hormone-binding globulin (SHBG) to a human prostatic cancer cell line (LNCaP) and the results of that binding were examined. Membranes derived from LNCaP cells bound unliganded SHBG on two sets of sites whose affinities were: Ka1 = 3.1 +/- 1.6 x 10(10) M-1 and Ka2 = 8.7 +/- 4.3 x 10(6) M-1. Intact cells also bound SHBG, but even after 6 h, less than 10% of specifically bound SHBG was internalized. This observation speaks against a role for the membrane binding of SHBG in steroid transport across cell membranes. When LNCaP cells were prebound with SHBG, addition of dihydrotestosterone or estradiol resulted in a dose-dependent increase in intracellular cAMP. SHBG in the absence of steroids or dihydrotestosterone in the absence of SHBG was without effect. 2-Methoxyestradiol, a steroid metabolite without biological activity, but which binds to SHBG more tightly than does estradiol, was also without effect. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.

2-Methoxyestradiol↗
PubMed1990

Plasma free and non-sex-hormone-binding-globulin-bound testosterone are decreased in obese men in proportion to their degree of obesity.

It is known that plasma total testosterone (T) is decreased in obese men in proportion to the degree of obesity, but similar information is not available for plasma free T and non-sex-hormone-binding globulin (SHBG)-bound T. We measured the 24-h mean plasma total T in 48 healthy (non-weight-stable men, aged 18-55 yr, with body mass indexes (BMI) ranging from 21-95 kg/m2. Free T and non-SHBG-bound T were calculated using the measured total T, the concentrations of albumin and SHBG, and the association constants of T to albumin and SHBG. Total body fat content was measured by deuterium-water isotope dilution. Findings were as follows. 1) BMI was very highly correlated with total body fat content (r = 0.96; P less than 0.001); thus, the degree of obesity can be calculated just as appropriately from simple height and weight measurements as from measurements of total body fat content. 2) Total, non-SHBG-bound, and free T were all highly correlated inversely with BMI; for total T, r = -0.727, P less than 0.01; for non-SHBG-bound T, r = 0.677, P less than 0.01; and for free T, r = -0.653, P less than 0.01. Thus, free T and non-SHBG-bound T are decreased in obese men in proportion to the degree of obesity, just as is the case for total T; percentage-wise, the decrease was the same for all 3 parameters.

Adult↗
PubMed1989

Solubilization and partial characterization of the sex hormone-binding globulin receptor from human prostate.

The sex hormone-binding globulin (SHBG) receptor was solubilized from the membranes of human prostate glands with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid). The binding activity of the soluble receptor was measured by allowing it to bind to 125I-SHBG and precipitating the complex with polyethylene glycol-8000. The binding activity was stable for at least 4 months at -20 degrees C and had a half-life of 23 days at 4 degrees C. Like the membrane-bound receptor, Scatchard analysis revealed two sets of binding sites for the soluble one. At equilibrium (24 h), the high affinity site had an association constant (KA) of 6.8 x 10(8) M-1 and a binding capacity of 1.4 pmol/mg protein, whereas the low affinity site had a KA of 4.7 x 10(6) M-1 and a binding capacity of 43 pmol/mg protein. At 37 degrees C, the association rate constant (k1) was 8.37 x 10(5) M-1 min-1 and the dissociation rate constant (k2) was 3.43 x 10(-4) min-1. The soluble receptor was retarded on Sepharose CL-6B and had an apparent Mr = 167,000.

Cholic Acids↗
PubMed1988

Induction of adenylate cyclase in a mammary carcinoma cell line by human corticosteroid-binding globulin.

Corticosteroid-Binding globulin (CBG) is a plasma protein that binds certain steroid hormones, mainly cortisol and progesterone. It has been demonstrated recently that specific binding sites for this protein exist on cell membranes. In this communication we establish that binding to these sites results in the induction of adenylate cyclase activity and the accumulation of cAMP in MCF-7 cells. These events are critically dependent upon a steroid being bound to CBG. These data are consistent with the hypothesis that CBG is a prohormone which is activated when cortisol is bound to it.

Adenylyl Cyclases↗
PubMed1988

Are corticosteroid-binding globulin and sex hormone-binding globulin hormones?

Because it no longer seemed reasonable to us that the sole function of the steroid-binding proteins in plasma was to serve as a buffer reservoir for steroid hormones, we conducted experiments which sought out other possibilities. Both CBG and SHBG bind to cell membranes, and this interaction partakes of the general characteristics of peptide hormone-membrane receptor systems. Additionally, human CBG has the ability to cause an increase in the activity of membrane-bound adenylate cyclase in MCF-7 cells, and this, in turn, results in an increase in cellular cAMP content. Thus, CBG appears to be a protein hormone. As a first consideration, one might presume that because CBG's half-life is measured in days, it would be counted among the hormones which, for the most part, are tonic in their effects, e.g., thyroid hormone. However, two important considerations tend to believe this presumption: (1) CBG which is unoccupied by steroid is not hormonally active (Figure 5): (2) Depending upon the time of day, circulating CBG is approximately 0-60% occupied in normal humans. These observations result in a circumstance in which a substantial portion of circulating CBG is available for activation by bursts of cortisol secretion. It seems prudent to speculate that, because steroids are essential for CBG's activity, the hormonal role of CBG may be entwined with, or complementary to the steroids which it binds. Finally, we should comment on the impact that our model of CBG as a hormone has on the view that only unbound steroid can be hormonally active. First, it should be stated that we have not addressed this question experimentally. Although there is evidence that CBG may be required for cortisol action, we feel that an obligate role for it is not documented adequately. At this time, we believe that CBG's hormonal role is compatible with a hypothesis that encompasses the view that unbound steroid hormones can diffuse into cells in some tissues and that both free and bound steroid can enter cells in others. Obviously, the final word on these important topics, as always, awaits the proper experiments.

Binding Sites↗
PubMed1988

Characteristics of the binding of corticosteroid-binding globulin to rat cell membranes.

Specific binding sites for corticosteroid-binding globulin (CBG) were detected on membranes prepared from rat spleen. The binding sites are typical of membrane receptors; they are saturable, specific, have high affinity, and require Mg2+ or Ca2+ for binding. There was little specific binding at 4 C, and maximal binding was obtained at 37 C. Scatchard analysis revealed a single set of binding sites with an apparent Kd of 0.84 microM, and a binding capacity of 39 pmol/mg membrane protein. The sites were specific for CBG; binding of [125I]CBG was not inhibited by a 10,000-fold excess of either rat albumin or rat transferrin. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the membrane-bound [125I]CBG revealed the presence of lower mol wt iodinated products which were undetectable in the unbound [125I]CBG fraction. Further, whereas the electrophoretic patterns from uterine, pulmonary, and renal membranes showed that the less mobile band (mol wt, 60K) of the normal CBG doublet appeared to be metabolized to a greater extent than the more mobile band (mol wt, 52K), those from splenic membranes showed equal metabolism of both parts of the doublet.

Animals↗