Corticosteroid-binding globulin (CBG) in fetal development

In fetal sheep the prepartum increase in plasma cortisol concentration is associated with an increase in high affinity corticosteroid binding activity in plasma. This appears to reflect an increase in corticosteroid-binding globulin (CBG) biosynthesis from the fetal liver, and evidence is presented that hepatic CBG gene expression is increased by exposure to glucocorticoids in the fetus. Immunoreactive CBG is found in other fetal tissues, and CBG mRNA is present in fetal pituitary. CBG reduces the ability of cortisol to exert negative feedback on basal or CRH-stimulated ACTH output by fetal sheep pituitary cells in culture. We suggest that CBG interacts with cortisol in a manner that maintains a low negative feedback on the pituitary, and perhaps hypothalamus. This constitutes a component of the cascade of events that is associated with hypothalamic-pituitary-adrenal activation in the late gestation fetus, and with the onset of parturition.     

Preparation of electrophoretic variants of corticosteroid-binding globulin (CBG) using liquid liquid partition chromatography

Human corticosteroid-binding globulin (CBG) was purified to homogeneity by application of three different chromatographic methods. After fractionation of pregnancy serum with ammonium sulfate the 80%-pellet was used for affinity chromatography based on tresyl activated Sepharose (Pharmacia, Uppsala, Sweden). The affinity eluate was injected into a Mono Q anion exchange column (Pharmacia). Fractions containing CBG were finally purified by liquid liquid chromatography on LiParGel 750 (Merck, Darmstadt, F.R.G.). The purified protein was characterized by IEF and PAGE. This paper describes a method for the chromatographic separation of the two variants of CBG without a loss of binding activity towards steroids for each of the two characteristic bands of this protein.     

Antogonistic effects of retinoic acid and triiodothyronine in the expression of corticoid-binding globulin (CBG) by cultured fetal hepatocytes

Cultures of rat fetal hepatocytes were used to investigate the effects and interplay of triiodothyronine (T₃) and retinoic acid (RA) in the regulation of gene expression of CBG, compared to that of -fetoprotein (AFP). The cultured cells showed cytological features typical to hepatocytes and actually synthesized CBG and AFP, as evidenced from in situ hybridization with specific radioactive and cell mRNA levels disappeared with a half-life of about 2 days, thereby reflecting the decrease previously seen in hepatic CBG mRNA contents during embryonic life. The K_d values for CBG binding were unchanged under these conditions. Culturing of hepatocytes in the presence of T₃ resulted in dose-dependent stimulations of both medium CBG and cell mRNA levels, with an EC₅₀ concentration of about 10⁻⁹ M. In sharp contrast, RA caused a reduction in CBG biosynthesis (IC₅₀ = 1.7 × 10⁻⁷ M) and, in addition, antagonized the stimulatory influence of T₃. Under the same experimental conditions, AFP synthesis failed to be affected in a similar fashion. We conclude that thyroid hormones and RA directly act on hepatocytes to specifically regulate the expression of CBG in an antagonistic way.     

Origin of corticosteroid-binding globulin in fetal rat. Comparative dynamics of corticosteroid-binding globulin, alpha-fetoprotein, and albumin secretion in primary cultures of fetal rat hepatocytes

The origin of corticosteroid-binding globulin (CBG) and its evolution in comparison with alpha-fetoprotein (AFP) and albumin synthesis, during early development of rat liver (days 13 and 15 of fetal life), have been investigated using cultured fetal hepatocytes. Synthesis and secretion of CBG, AFP, and albumin is evidence by cycloheximide-sensitive [14C]leucine incorporation into immunoprecipitable polypeptides secreted by cultured hepatocytes into the medium, two-dimensional immunoelectrophoretic and autoradiographic identification of newly synthesized labeled proteins, corticosterone and estradiol-17 beta binding to CBG and AFP, respectively, and indirect immunofluorescence localization of AFP, albumin, and CBG in cultured fetal hepatocytes. CBG, albumin, and AFP accounted for 6, 11, and 25% (in 13-day-old rat fetuses) and 5, 15, and 28% (15-day-old rat fetuses), respectively, of the total secreted proteins in the culture medium. The rates of CBG, AFP, and albumin (counts/minute of secretion [14C]leucine incorporated per milligram of cell protein/hour of culture) in the hepatocytes of 15-day-old rat fetuses were 1.48-, 2.1-, and 2.57-fold higher, respectively, than in the 13-day-old rat fetuses. These results indicate that fetal liver is also active in CBG synthesis, along with AFP and albumin, as early as day 13 of fetal life and that the synthetic rates of these secretory proteins depend upon the developmental stage of the fetal liver. This developmental related change in the rate of synthesis of CBG by the fetal hepatocytes may regulate the level of free (active) glucocorticoid in the fetal circulation and thereby the initiation and regulation of glucocorticoid-dependent processes during the crucial stages of the differentiation of fetal liver and other developing tissues.     

Rabbit corticosteroid-binding globulin: primary structure and biosynthesis during pregnancy

The cDNA-deduced primary structure of rabbit corticosteroid-binding globulin (CBG) contains 383 amino acids (mol wt, 42,326), including three cysteine residues and four sites for N-glycosylation. It is primarily the product of a 1.68-kilobase hepatic mRNA, but small amounts of CBG mRNA were also found in maternal lung, spleen, and ovary and fetal kidney. In the fetus, hepatic CBG mRNA concentrations increase markedly after day 11 and were 2- to 5-fold higher than those in maternal liver during days 17-23. They then declined to very low levels at term (31 days). By contrast, maternal hepatic CBG mRNA levels did not increase until day 23; reached a peak at about day 27, and then declined to prepregnancy values by 3 days after delivery. In general, fetal and maternal hepatic CBG mRNA concentrations reflect the corresponding serum CBG levels. Our data, therefore, indicate that the marked changes in fetal and maternal plasma CBG levels during pregnancy reflect changes in the biosynthesis of the protein rather than alterations in compartmentalization or clearance.     

Progesterone measurement using stripped corticosteroid binding globulin (CBG)

Modifications in an existing competitive protein binding assay for progesterone have been made which provide a readily available and rich source of binding sites on the corticosteroid binding globulin (CBG). The primary and most important modification is the rapid removal of endogenous steroids from plasma by gel filtration at an elevated temperature. The ‘stripped’ protein retains full CBG activity, but is cleared of 95% of its endogenous steroids. This stripping procedure provides not only increased number of binding sites, but in conjunction with the other modifications also eliminates some of the variability in the assay.     

Alterations in sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), and steroid hormone concentrations during pregnancy in rhesus macaques

Temporal relationships between concentrations of sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), total and free estradiol, total and free testosterone, cortisol, and progesterone were studied in plasma obtained at 1- to 3-day intervals throughout gestation in six rhesus macaques. Concentrations of SBP and CBG were measured by diethylaminoethyl cellulose filter assays. Total and free steroids were estimated by radioimmunoassay and ultrafiltration dialysis, respectively. We found that SBP was elevated between days 30 and 50 and CBG between days 60 and 140; both then declined until term (167 days). Estradiol increased gradually throughout gestation. Testosterone was elevated between days 10 and 40, then declined, and rose slightly in late gestation until approximately 15 days before delivery, when it increased markedly. Free estradiol and testosterone increased dramatically before parturition. Progesterone was elevated between days 25 and 45 and declined to relatively constant levels thereafter. Cortisol was essentially unchanged throughout gestation. Our data show that in the pregnant rhesus, levels of SBP and CBG vary independently of one another, but both decline before term; concentrations of both total and free estradiol and testosterone increase markedly before parturition; in late gestation, elevated estrogen is not associated with increased levels of SBP or CBG (as it is in human females).     

An enzyme-linked immunosorbent assay (ELISA) for human corticosteroid-binding globulin

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for human corticosteroid-binding globulin was developed. A polyclonal rabbit anti-CBG antibody is immobilised to a microtitre plate. Following incubation of standards and samples a second monospecific rabbit anti-CBG antibody, labelled with alkaline phosphatase, is added. After colour development the microtitre plate is read at 405 nm wavelength. The assay shows good agreement to CBG binding capacity assay and commercially available RIA.     

Plasma cortisol and corticosteroid-binding globulin in essential hypertension

Plasma concentration of cortisol, total CBG-binding capacity, and blood pressure were measured in control subjects (n = 171), patients with essential hypertension (EH; n = 210) and their first-degree normotensive (NR; n = 84) or hypertensive (HR; n = 66) relatives. Mean (+/- SD) plasma cortisol was significantly (p less than 0.001) decreased in EH (10.1 +/- 4.3 g/dl) patients and HR (11.7 +/- 4.1). Plasma cortisol in NR did not differ from control values (14.3 +/- 4.5) but the distribution of individual values covered the entire control-EH (14.6 +/- 5.5) range. Mean (+/- SD) CBG-binding capacity was significantly (p less than 0.001) lower in EH (14.4 +/- 3.0), NR (17.5 +/- 2), HR (17.6 +/- 2.2) as compared to controls (20.9 +/- 2.1), indicating that the decline in EH and in most relatives was mainly in plasma CBG-bound cortisol. The plasma CBG-binding capacity for cortisol was significantly negatively correlated with mean arterial pressure (MAP) in both controls (p less than 0.001) and NR (p less than 0.01) but not in either HR (r = 0.02) or never-treated EH patients. Total afternoon plasma aldosterone was higher (p less than 0.01 vs. controls) in 93 untreated EH patients (11.2 +/- 4.8 ng/dl) than in either 161 first-degree relatives (8.1 +/- 3.4 ng/dl) or 117 controls (7.6 +/- 3.5 ng/dl). The respective aldosterone-binding globulin (ABG) binding capacities for aldosterone were 21.2 +/- 6.7, 20.1 +/- 9.3 and 9.8 +/- 4.0%. In all these subjects taken together, there was a positive correlation between MAP and ABG-binding capacity (r = 51; p less than 0.001). The association of reduced plasma cortisol and decreased CBG binding capacity in EH may be closely related to altered steroid metabolism, which may be partly explained by an abnormality resembling a relative deficiency in adrenal 17 alpha- and 11 beta-hydroxylation. In some EH patients, hypertension may be the result of the ineffectiveness of plasma cortisol in preventing slightly elevated endogenous ACTH levels leading to an increase in ACTH-sensitive steroids.     

Perfusion culture of fetal human hepatocytes in microfluidic environments

Various types of bioreactors composed of microstructured PDMS (Polydimethylsiloxane) layers have recently been fabricated for perfusion culture of mammalian cells such as adult rat hepatocytes. As a new feature of those bioreactors, in this study, cultivation of fetal human hepatocytes (FHHs) was attempted, because they have high possibility to mature in vitro with preserving their normality, which is suitable for inplantation of liver tissue equivalents reconstituted in vitro. During the perfusion culture in the PDMS bioreactors for 1 week, cells showed good attachment, spreading and reached their confluence over the channels. In addition, their albumin production was significantly enhanced in the perfusion culture using the PDMS bioreactors up to about four times during the FHH perfusion culture when compared in dish-level static culture. Hep G2 cell cultures were also performed and have also shown under perfusion conditions an enhanced cell activity multiplied by 2 compared to static conditions. Although, the cellular activities of FHH cells are still low even compared to those of the Hep G2 cells, the conclusions of this work is encouraging toward future liver tissue engineering based on in vitro propagation and maturation of hepatocyte progenitors combined with microfabrication technologies.     

The effect of dexamethasone on albumin production by fetal rat hepatocytes in culture

Hepatocytes derived from 15 and 19-day gestation rats synthesize and secrete albumin during culture. Albumin secretion is maintained when the culture medium is supplemented with dexamethasone but declines in its absence. The fall in secretion rate correlates with the level of albumin messenger RNA in the respective cultures. Even when dexamethasone is present, the level of albumin production in 19-day gestation hepatocytes is 6 to 7 times greater than that observed in hepatocytes derived from 15-day gestation rats. Immunocytochemical studies were undertaken to establish whether the difference in secretion rate was due to a difference in the amount of albumin produced by all the hepatocytes of the respective cultures or whether there were fewer hepatocytes which were capable of synthesizing albumin in the less mature liver. The results indicate that albumin production is reduced in all hepatocytes when cultured in the absence of dexamethasone.     

Regulation of plasma corticosteroid-binding globulin in adult cynomolgus monkey (Macaca fascicularis) during different reproductive states

The plasma concentration of the corticosteroid-binding globulin (mCBG) has been measured in Macaca fascicularis, during different stages of reproduction and under hormonal treatments. The mCBG level was determined by a specific electroimmunoassay. There was no difference between females in the follicular phase and intact males; mCBG concentrations were respectively (mean +/- SEM) 469 +/- 53 and 443 +/- 25.6 nmol/l. The mCBG levels levels were similar during both the luteal (469 +/- 33.5 nmol/l) and the follicular phase (469 +/- 53 nmol/l). Compared to intact males, the mCBG levels were higher (P less than 0.05) in castrated males (527 +/- 6.6 nmol/l). During gestation, no systematic variations were found and the mCBG levels were not statistically different from the values found during the follicular phase. When estradiol benzoate was administered to castrated animals, the mCBG concentrations increased rapidly. In contrast, the values were reduced slightly by testosterone treatment. The sex-steroid action on the mCBG levels was discussed and compared with the mSBP levels. We question also, the mechanisms involved in the regulation of the mCBG levels during pregnancy.     

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.     

Influence of ornithine on albumin synthesis by fetal and neonatal hepatocytes maintained in culture

Fetal rat as well as fetal and neonatal human hepatocytes were cultured in an arginine-free medium with or without L-ornithine. This amino acid was found greatly to improve cell survival of both rat and human parenchymal cells and to favour their proliferation. In addition, L-ornithine appeared to modulate the expression of various functions and particularly to increase albumin secretion. Since a correlated increase of the corresponding mRNA was observed, it may be postulated that L-ornithine acts at a pre-translational level.