Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus).

Polar bears (Ursus maritimus) from several subpopulations undergo extended fasting during the ice-free season. However, the animals appear to conserve protein despite the prolonged fasting, though the mechanisms involved are poorly understood. We hypothesized that elevated concentrations of corticosteroid binding globulin (CBG), the primary cortisol binding protein in circulation, lead to cortisol resistance and provide a mechanism for protein conservation during extended fasting. The metabolic state (feeding vs. fasting) of 16 field sampled male polar bears was determined based on their serum urea to creatinine ratio (>25 for feeding vs. <5 for fasting). There were no significant differences in serum cortisol levels between all male and female polar bears sampled. Serum CBG expression was greater in lactating females relative to non-lactating females and males. CBG expression was significantly higher in fasting males when compared to non-fasting males. This leads us to suggest that CBG expression may serve as a mechanism to conserve protein during extended fasting in polar bears by reducing systemic free cortisol concentrations. This was further supported by a lower serum glucose concentration in the fasting bears. As well, a lack of an enhanced adrenocortical response to acute capture stress supports our hypothesis that chronic hunger is not a stressor in this species. Overall, our results suggest that elevated serum CBG expression may be an important adaptation to spare proteins by limiting cortisol bioavailability during extended fasting in polar bears.

Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

Corticosteroid-Binding Globulin is expressed in the adrenal gland and its absence impairs corticosterone synthesis and secretion in a sex-dependent manner.

Corticosteroid-binding globulin (CBG) is synthesized by the liver and secreted into the bloodstream where binds to glucocorticoids. Thus CBG has the role of glucocorticoid transport and free hormone control. In addition, CBG has been detected in some extrahepatic tissues without a known role. CBG-deficient mice show decreased total corticosterone levels with missing of classical sexual dimorphism, increased free corticosterone, higher adrenal gland size and altered HPA axis response to stress. Our aim was to ascertain whether CBG deficiency could affect the endocrine synthetic activity of adrenal gland and if the adrenal gland produces CBG. We determined the expression in adrenal gland of proteins involved in the cholesterol uptake and its transport to mitochondria and the main enzymes involved in the corticosterone, aldosterone and catecholamine synthesis. The results showed that CBG is synthesized in the adrenal gland. CBG-deficiency reduced the expression of ACTH receptor, SRB1 and the main genes involved in the adrenal hormones synthesis, stronger in females resulting in the loss of sexual dimorphism in corticosteroid adrenal synthesis, despite corticosterone content in adrenal glands from CBG-deficient females was similar to wildtype ones. In conclusion, these results point to an unexplored and relevant role of CBG in the adrenal gland functionality related to corticosterone production and release.

Synthesis and secretion of corticosteroid-binding globulin by rat liver. A source of heterogeneity of hepatic corticosteroid-binders.

Classical glucocorticoid receptors (type II) have a high affinity for synthetic and natural glucocorticoids. We have previously demonstrated an additional binding site in kidney cytosol (type III) which has a high affinity for corticosterone but a low affinity for dexamethasone. In many ways, this binder resembles plasma corticosteroid-binding globulin (CBG). The first goal of this study was to determine the organ distribution of the type III binding sites. Cytosol was prepared from isolated cells to avoid plasma contamination. Of the tissues examined, type III sites were found only in liver and kidney; sites were absent from thymocytes, IM-9 lymphocytes, adipocytes, and bone cells. The second goal of this study was to ascertain whether CBG is synthesized in liver and kidney. Liver and kidney slices were incubated in vitro and the concentration of type III sites was seen to rise in hepatic cytosol and incubating medium but not kidney. To verify the impression that liver was synthesizing and secreting CBG, the following experiments were performed: (a) To demonstrate that type III sites were CBG, steroid-binding profiles and migration on polyacrylamide gel electrophoresis were shown to be identical for hepatic type III sites and serum. (b) To indicate that the rise in type III sites was dependent on protein synthesis, it was shown that cycloheximide blocked the appearance of new type III sites. (c) To establish that the type III sites were being secreted, in situ liver perfusion experiments showed time-dependent release of new sites into the perfusate. In conclusion, liver synthesizes and secretes type III sites, a finding previously suspected but never proved. The presence of type III sites in kidney remains to be explained.

Expression of corticosteroid binding globulin in the rat central nervous system.

Immunoreactivity for corticosteroid binding globulin was observed in the hypothalamus of intact male rats in the magnocellular nuclei and in single neurons in the periventricular nucleus and the lateral hypothalamus. The suprachiasmatic and the arcuate nuclei contained parvocellular neurons with specific immunoreactivity. Extensive networks of immunopositive fibers were observed in the lateral hypothalamus, the preoptic region, the bed nucleus of the stria terminalis and along the third ventricle. Immunostained axons often exhibited varicosities. The internal and the external layer of the median eminence showed numerous bundles of immunostained axons. Herring bodies in the posterior pituitary lobe contained specific immunoreactivity while pituicytes remained unstained. A portion of the Purkinje cells in the cerebellum and mossy fibers in the cerebellar granular layer stained for corticosteroid binding globulin. Some of the pyramidal cells in the hippocampus were corticosteroid binding globulin positive. Immunostained fibers occurred in the mesencephalon in the periaqueductal grey and in the medulla oblongata. A small fraction of the ependymal cells was also stained. In the spinal cord we observed specific immunoreactivity in a portion of the neurons in the dorsal horn. With polymerase chain reaction we confirmed the presence of the respective transcripts in the different brain regions. The multiple locations of corticosteroid binding globulin throughout the central nervous system suggest multiple functional properties, including neuroendocrine and neurohumoral functions.

Hormonal effects on the secretion and glycoform profile of corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) is a plasma glycoprotein that is primarily synthesized in the liver and binds cortisol and progesterone with high affinity. In this study, a CBG secreting hepatocellular carcinoma derived cell line (HepG2) was used to investigate the hormonal regulation of hepatic CBG synthesis. HepG2 cells were grown for 72 h in 30, 300 and 3000 nM concentrations of estradiol (E2), testosterone (T), insulin, thyroxin (T4) and dexamethasone (DMZ) and the secreted CBG quantified by a novel enzyme-linked immunosorbent assay (ELISA). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was carried out to determine the effects of these hormones on the relative distribution of CBG glycoforms. Insulin, T4 and high concentrations of E2 decreased the secretion of CBG by HepG2 cells (p<0.05). Ethanol, the solvent used for E2, T and DMZ, also significantly attenuated CBG secretion. 2D-PAGE resolved 13-14 glycoforms of CBG produced by HepG2 cells. Insulin caused a reduction in the synthesis of more acidic, while T4 and DMZ decreased the production of more basic CBG glycoforms. Stimulation with E2 resulted in the synthesis of additional isoforms of increased acidity, which may represent a type of CBG only seen during pregnancy in vivo. Possible physiological implications of these findings are discussed.

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.

Interleukin-6 inhibits corticosteroid-binding globulin synthesis by human hepatoblastoma-derived (Hep G2) cells.

Corticosteroid-binding globulin (CBG) belongs to the superfamily of serine proteinase inhibitors which include alpha 1-antitrypsin, alpha 1-antichymotrypsin, and T4-binding globulin. Interleukin-6 (IL-6), the main mediator of the acute phase phenomenon, increases alpha 1-antitrypsin and alpha 1-antichymotrypsin synthesis and decreases T4-binding globulin synthesis by human hepatoblastoma-derived (Hep G2) cells. This effect is predominantly at a transcriptional level. When Hep G2 cells were exposed to different concentrations of IL-6 for variable time intervals, IL-6 caused a dose- and time-dependent decrease in the amount of [35S]methionine-labeled CBG immunoprecipitated in the culture medium. This effect could be greatly reduced by preincubation of IL-6 with its neutralizing antibody and reversed by removing the cytokine from the culture medium. The secretion rate of CBG was not affected by cell exposure to IL-6. CBG mRNA steady state levels were reduced; changes in mRNA were quantitatively similar to changes in secreted protein. Nuclear run-off assays failed to show a change in the rate of transcription of the CBG gene. These data indicate that IL-6 diminishes CBG synthesis by Hep G2 cells acting at a posttranscriptional level, presumably through a reduced stability of mRNA. In view of the role of IL-6 in the inflammatory process and other acute phase phenomena, these data suggest that its effects on CBG synthesis might influence the bioavailability of cortisol indirectly and play a role in regulating the homeostatic process during these conditions.

Synthesis in vitro of corticosteroid-binding globulin from rat liver messenger ribonucleic acid.

To show that corticosteroid-binding globulin (CBG), a plasma glycoprotein, is synthesized in the liver as well as to develop a tool with which to study the hormonal factors which control its synthesis, we undertook to translate rat CBG mRNA in vitro. Rat liver mRNA was translated in the presence of [35S]methionine in both a rabbit reticulocyte lysate (cell-free) System and a Xenopus laevis oocyte (whole cell) system. A monospecific antibody against highly purified rat CBG was used to precipitate a single 35S-labeled product from the mixture of newly synthesized hepatic proteins in the two systems. In the rabbit reticulocyte lysate system, the protein migrated with an apparent molecular weight of 41,000 on sodium dodecyl sulfate-gel electrophoresis; CBG isolated from rat serum migrated as a doublet with apparent molecular weights of 56,000 and 62,500. Since proteins synthesized by a cell-free system are not glycosylated, the difference in molecular weight can probably be accounted for by the carbohydrate content of plasma CBG. Translation of rat liver mRNA in a Xenopus laevis oocyte system again resulted in immunoprecipitation of a single 35S-labeled protein, but this time with an apparent molecular weight of 53,000 on sodium dodecyl sulfate-gel electrophoresis; this probably represents glycosylated CBG and corresponds to the apparent molecular weight of one of the species isolated from rat plasma.

Plasma adrenocorticotropin is more sensitive than transcortin production or thymus weight to inhibition by corticosterone in rats.

After adrenalectomy, ACTH, corticosterone-binding globulin (CBG), and thymus wet weight increase in rats as a consequence of the removal of corticosterone (B) and decrease again in response to replacement with glucocorticoids. We have studied the effect of replacing adrenalectomized male rats with a variety of different concentrations of B. Plasma concentrations of ACTH and CBG and thymus wet weights were related to the measured concentration of free ultrafilterable B in plasma. In other experiments, the clearance of [125I]CBG was determined in adrenalectomized rats with and without B replacement, and the time required for the changes in plasma CBG concentrations after removal and/or replacement of B in adrenalectomized rats was determined. Five to 7 days after adrenalectomy and institution of a relatively constant B replacement signal, plasma CBG concentrations were highly correlated with circulating B concentrations (r2 = 0.745; P less than 0.001). The effect of B was on the CBG production rate, since clearance did not change. Because CBG concentrations decrease as total B concentrations increase, there is an amplification of free B concentrations with increasing total B. The relationships of plasma ACTH and CBG and thymus wet weight to circulating free B levels showed that 50% inhibition of ACTH was achieved at a free B concentration of 0.8 +/- 0.05 nM, whereas 50% inhibition of CBG and thymus wet weight were achieved at free B concentrations of 4.6 +/- 0.9 and 4.4 +/- 0.6 nM, respectively. These inhibition values correlate well with the known Kd values for the high affinity type I B receptor (0.5 nM) and for the lower affinity type II glucocorticoid receptor (2.5-5 nM), respectively, suggesting that ACTH secretion, CBG production, and thymus wet weight are regulated by the association of B with these receptor types.

Studies on the role of glycosylation for human corticosteroid-binding globulin: comparison with that for thyroxine-binding globulin.

The role of glycosylation on the secretion and the stability of human corticosteroid binding globulin (CBG) was studied. Cells of the human hepatoma line were labeled by [35S]methionine in presence of or absence of tunicamycin (TM). Media or cells were harvested at 0, 3, 6, and 20 h after the addition of excess unlabeled methionine. Media and cell lysates were incubated with anti-CBG serum and immune complexes were precipitated with Staphylococcus aureus protein A (Pansorbin). Immunoprecipitates were analyzed by fluorography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitation of T4-binding globulin (TBG) was also carried out with anti-TBG serum. Fluorographic analysis revealed three forms of CBG: CBG1, a glycosylated, mature, and secretory form with apparent mol wt of 70 K; CBG2, a glycosylated precursor which due to incomplete carbohydrate processing has an apparent mol wt of 54 K; and CBG3, a nonglycosylated form consisting of the 40 K core protein. In absence of TM, CBG1 was observed in media and CBG2 was detected in cell lysates. The proportion of CBG1 increased during the chase, whereas that of CBG2 decreased, indicating that CBG was secreted after processing of the oligosaccharides on CBG2. In presence of TM, CBG3 was found both in media and cell lysates. The sum of CBG3 in the medium and the cell lysate decreased during the chase, whereas that of CBG1 and CBG2 remained unchanged. Similar to CBG, TBG1 (mature form, 60 K) and TBG2 (partially processed glycosylated form, 54 K) were observed in media and cell lysates, respectively, in absence of TM. However, TBG3 (nonglycosylated, 44 K) was not detected in medium. These results indicate that glycosylation is not a key factor for the secretion of CBG but is important for its stability. On the other hand the glycosylation is indispensable for the secretion of TBG.

Squirrel monkey corticosteroid-binding globulin: primary structure and comparison with the human protein.

Squirrel monkey (Saimiri sciureus) corticosteroid-binding globulin (CBG) is the product of a 1.6-kilobase mRNA in the liver. Analyses of two overlapping cDNAs revealed that the squirrel monkey CBG precursor comprises 406 amino acids, the first 22 residues of which exhibit 91% identity with the human CBG leader sequence. The mature form of squirrel monkey CBG, therefore, very likely comprises 384 amino acids and has a polypeptide mol wt of 42,854. Compared to human CBG, the squirrel monkey protein contains an additional residue (threonine) at position 144, and the two proteins exhibit 86% sequence identity if this is taken into account. Squirrel monkey CBG contains five consensus sites for N-glycosylation, four of which are located in analogous positions in human CBG, and has two cysteine residues in the same relative positions as the cysteines in human CBG. Unlike CBG in most other species, squirrel monkey CBG appears to circulate as a dimer, and its affinity for glucocorticoids is remarkably low. We, therefore, expressed cDNAs for human and squirrel monkey CBGs in Chinese hamster ovary (CHO) cells and compared the physico-chemical properties of the products with those of the corresponding serum proteins. Squirrel monkey CBG is produced by CHO cells as a dimer, and its subunit size heterogeneity is similar to that associated with CBG in serum. In addition, the cortisol-binding affinity of squirrel monkey CBG produced by CHO cells is similar to that of the natural protein and is 5- to 8-fold lower than that of natural or recombinant human CBG. Mutants in which a threonine at position 144 was either added to human CBG or subtracted from squirrel monkey CBG were also expressed in CHO cells. This demonstrated that this additional amino acid in the squirrel monkey CBG sequence may actively contribute to its propensity for spontaneous dimerization, but does not account for its relatively low steroid-binding affinity.

Postnatal development of corticosteroid-binding globulin: effects of thyroxine.

The aim of this study was to examine the temporal and dose characteristics of the corticosteroid-binding globulin (CBG) response to T4 and to determine whether this response is due to stimulation of hepatic biosynthesis of CBG. When n-propylthiouracil (PTU)-induced hypothyroid pups were given a single injection of T4 (0.1 microgram/g BW) on postnatal day 5, 6, or 7, only pups treated on day 7 showed a significant increase in CBG. In a T4 dose-response study conducted with 5- and 8-day-old pups, older pups exhibited maximum CBG concentrations (Rmax) which were 2.5-fold higher than those of younger pups. The D1/2 (dose required to elicit half the maximum response) values were similar at both ages. The effect of T4 withdrawal on serum CBG was also studied in PTU-treated pups. T4 injection on postnatal days 5-19 resulted in a progressive rise in CBG. In pups treated with T4 on days 5-9 and then withdrawn from treatment through day 20, serum CBG showed no further increase but was maintained at an elevated level. Using a liver slice system to assess CBG production in vitro, livers from 14-day-old hyperthyroid pups produced 4.77 ng corticosterone bound/g liver, while livers from euthyroid pups produced no CBG. We conclude: 1) the response of CBG to T4 is a function of the age of the animal; between days 5 and 8 this is due to increased Rmax without any change in sensitivity to T4 (D1/2); 2) T4 is required not only to initiate but also to sustain the developmental increase in CBG; and 3) T4 elicits an increase in circulating CBG by stimulating its synthesis.

Hormonal regulation of corticosteroid-binding globulin biosynthesis in the male rat.

Corticosteroid-binding globulin (CBG) is an acidic glycoprotein produced primarily by the liver and is the major glucocorticoid transport protein in rat blood. Various hormone treatments are known to modify plasma CBG levels and may thereby influence the bioavailability of glucocorticoids. We have, therefore, examined serum CBG and hepatic CBG mRNA levels in male rats after sc steroid (25 micrograms/100 g BW twice daily of dexamethasone, prednisolone, corticosterone, estrone, estradiol, ethinyl estradiol, progesterone, or 5 alpha-dihydrotestosterone) or T4 (10 micrograms/100 g BW twice daily) administration. After dexamethasone treatment, serum CBG levels decreased significantly (P less than 0.002) within 48 h and were 26% of those in vehicle-treated animals by 72 h. At this time, CBG mRNA was undetectable in liver RNA extracts by Northern blotting or solution hybridization, and nuclear run-off experiments indicated that dexamethasone reduces CBG gene transcription in the liver by at least 14-fold. Treatment with ethinyl estradiol also reduced serum CBG levels significantly (P less than 0.015) compared to pretreatment values. No other significant changes in serum CBG levels were observed after any other steroid hormone treatment, and alterations in hepatic CBG mRNA levels were only observed after dexamethasone treatment. T4 administration for 72 h increased serum CBG and hepatic CBG mRNA levels by 66% and 39%, respectively, but did not influence the rate of CBG gene transcription. Thus, increased CBG biosynthesis after thyroid hormone treatment appears to be due to an increase in CBG mRNA stability. No change in the Concanavalin-A-binding properties of CBG molecules was observed after treatment with either dexamethasone or T4, and this suggests that these hormones do not influence the type of oligosaccharide chains associated with this protein. We, therefore, conclude that dexamethasone and T4 regulate hepatic CBG biosynthesis in the rat by modulating CBG mRNA synthesis and stability, respectively.

Corticosteroid-binding globulin biosynthesis in the mouse liver and kidney during postnatal development.

Plasma corticosteroid-binding globulin (CBG) is produced by the liver, but low levels of CBG mRNA have been detected in other tissues, including the kidney. Glucocorticoids influence postnatal renal development in rodents, and CBG production in the kidney may influence the local bioavailability of glucocorticoids. We, therefore, used in situ hybridization and immunohistochemistry to define the sites of CBG biosynthesis during postnatal development and have found that the liver and kidney are major sites of CBG biosynthesis in the first weeks of life. Both CBG and its mRNA were undetectable in the neonatal liver, and only a weak hybridization signal for CBG mRNA was present in the 7-day-old mouse liver. In neonatal mice, the developing tubules of the kidney represent the most active site of CBG biosynthesis, and immunoreactive CBG was also detected in the same cells. By 7 days of age, CBG and its mRNA were colocalized to the proximal convoluted tubules of the juxtamedullary nephrons. The abundance of CBG mRNA in the liver increased from 10 days of age and was accompanied by similar increases in serum CBG until adult levels were reached by 4 weeks of age. In contrast, CBG mRNA in the kidney increased to a maximum during the third week of life, but was undetectable 3 weeks later. The CBG within the proximal convoluted tubules was located in secretory granules close to the luminal surface of the epithelial cells, suggesting that it is secreted into the tubular lumen. Western analysis revealed that marked proteolytic degradation of CBG occurs in the urine concurrently with an increase in CBG biosynthesis in the developing kidney. Thus, the liver is not the only site of CBG biosynthesis in the developing mouse, and CBG production by the epithelial cells of the proximal convoluted tubules may influence glucocorticoid-dependent maturation of the kidney tubules by a process that somehow involves proteolytic degradation.

Developmental regulation of corticosteroid-binding globulin biosynthesis in the baboon fetus.

The present study determined the roles of estrogen and cortisol in maternal and fetal corticosteroid-binding globulin (CBG) levels and fetal hepatic messenger RNA (mRNA) expression in the baboon. Samples of fetal liver, kidney, and brain were obtained from untreated control animals at early (day 60; n = 4), mid (day 100; n = 8), and late (day 165; n = 5) gestation (term = day 184). Maternal and umbilical blood samples were collected on day 100 from baboons in which betamethasone was administered sc to the mother (n = 6) on days 60-99 of gestation and on day 165 from animals (n = 4) in which the fetus was administered betamethasone on days 150-164 of gestation. Maternal serum cortisol concentrations were similar at mid (43 +/- 2 micrograms/dl) and late (42 +/- 3 micrograms/dl) gestation and decreased (P < 0.05) at midgestation (1 +/- 1 micrograms/dl) and term (31 +/- 4 micrograms/dl) after betamethasone treatment. Umbilical serum cortisol levels were also reduced (P < 0.05) at both mid (1 +/- 1 micrograms/dl) and late (14 +/- 5 micrograms/dl) gestation by betamethasone treatment. Fetal serum CBG levels in untreated animals were lower (P < 0.05) on day 165 (444 +/- 29 pmol/ml) than on day 100 (844 +/- 35 pmol/ml) and increased (P < 0.05) at midgestation (1098 +/- 64 pmol/ml), but not at term (551 +/- 24 pmol/ml), after betamethasone treatment. In contrast, maternal serum CBG levels (range, 528-770 pmol/ml) were not altered by gestational age or betamethasone. The human CBG complementary DNA hybridized to a single mRNA species of 1.8 kilobases in baboon fetal liver; however, CBG was not expressed in fetal kidney and was detectable in fetal brain and pancreas only by reverse transcription-PCR. In untreated baboon fetuses, the mRNA levels of hepatic CBG, expressed as a ratio of 18S RNA, progressively decreased (P < 0.05) in early (1.83 +/- 0.17), mid (0.97 +/- 0.12), and late (0.51 +/- 0.04) gestation. These results demonstrate that fetal hepatic CBG mRNA expression and serum CBG concentrations were elevated early in baboon gestation and exhibited a progressive decline during the course of advancing pregnancy. We suggest that the increased levels of fetal CBG in the early stages of gestation reflect stimulation of hepatic CBG synthesis by maternal cortisol, which we previously demonstrated to occur in the fetus as a result of preferential 11 beta-hydroxysteroid dehydrogenase-catalyzed glucocorticoid reduction across the placenta. The decline in fetal CBG may reflect the developmental increase in catabolism of cortisol to bioinactive cortisone in target tissues of the fetus such as the liver.

Effect of adrenocorticotropin administration on the biosynthesis of corticosteroid-binding globulin in fetal sheep.

Parturition in sheep is initiated by the fetus through activation of the fetal hypothalamic-pituitary-adrenal axis and is associated with increased concentrations of ACTH, cortisol, and corticosteroid-binding globulin (CBG) in the fetal circulation during the final 10-15 days of pregnancy. Premature parturition and a precocious elevation in fetal plasma CBG are produced by intrafetal ACTH administration, but the possible sources of CBG in the ovine fetus are not known. To determine these sites, CBG mRNA was measured in tissues from fetal sheep in late pregnancy and after intrafetal ACTH treatment, using a sheep CBG cDNA. Fetal ACTH treatment caused a significant increase in the fetal plasma corticosteroid-binding capacity (CBC), although there was no significant difference in CBC between umbilical arterial and umbilical venous plasma. After ACTH treatment, CBC was elevated in fetal liver and kidney. Cortisol binding in these tissues had characteristics similar to those of cortisol binding in fetal sheep plasma. By Northern blot analysis a single mRNA (1.7 kilobases) for CBG was detected in fetal liver, kidney, lung, and adrenal, but not in placenta. The abundance of CBG mRNA in the fetal liver was greater than that in other tissues, but was unchanged by ACTH treatment. The level of CBG mRNA in the fetal kidney, but not in other tissues, increased 3-fold after ACTH. We conclude that the elevation in plasma CBC after intrafetal ACTH, and presumably also at term pregnancy, does not reflect production of CBC by the placenta or transfer from the mother. Rather, it results from production primarily in the fetal liver and kidney, although only in the latter tissue is CBG mRNA accumulation increased by intrafetal ACTH treatment.

Characterization of pulsatile secretion and clearance of plasma cortisol in premature and term neonates using deconvolution analysis.

Pulsatile secretion of cortisol (F) has not been documented in the newborn infant. Using repeated blood sampling and deconvolution analysis, we investigated F secretion and elimination dynamics in a group of five premature (gestational age, 24-34 weeks) and five term neonates. These infants had required placement of an umbilical arterial cannula for monitoring respiratory status, but were otherwise clinically stable. Blood samples were obtained at 15-min intervals for a 6-h period. All plasma F determinations were 58 nmol/L (2.1 micrograms/dL) or more, and pulsatile F secretion was observed in all infants. No significant differences were noted between the two groups with regard to 6-h mean plasma F concentration [350 +/- 129 (premature) vs. 277 +/- 54 nmol/L (term)], plasma corticosteroid-binding globulin (14 +/- 0 vs. 13 +/- 1 mg/L), F secretory burst frequency (4 +/- 0 vs. 5 +/- 1 bursts/6 h), mass of F secreted per burst [760 +/- 480 vs. 310 +/- 100 nmol/Lv [Lv, liter of F distribution volume)], F production rate (FPR; 2.7 +/- 1.4 vs. 1.1 +/- 0.2 mumol/Lv.6 h), or plasma F half-life (45 +/- 6 vs. 56 +/- 4 min). However, the premature infants had a significantly longer F secretory burst half-duration (63 +/- 18 vs. 6.7 +/- 4.0 min; P < 0.01) and a significantly lower maximal F secretory rate (9.4 +/- 3.4 vs. 100 +/- 26 nmol/Lv.min; P < 0.02) than the term infants. Body surface area and body weight were inversely correlated with F secretory burst half-duration (r = -0.74 and -0.75, respectively); both were also positively correlated with the maximal F secretory rate (r = 0.66 and 0.72). The two most premature infants had significantly greater mean plasma F and FPR than the other three premature and all of the term infants. Extrapolating to 24 h and correcting for the distribution volume of F and body surface area, we estimate FPR to be approximately 17-24 mumol/m2.24 h (6.6-8.8 mg/m2.24 h) for newborn infants of 34 weeks or more gestational age. These values are consistent with newer estimates of FPR in older children and adults determined using either deconvolution analysis or stable isotope dilution methods.

Corticosteroid-binding globulin (CBG) production by hepatic and extra-hepatic sites in the ovine fetus; effects of CBG on glucocorticoid negative feedback on pituitary cells in vitro.

Plasma cortisol levels increase in fetal sheep during late gestation and this is associated with an increase in plasma corticosteroid-binding globulin (CBG) concentrations. However, the relative tissue sources of plasma CBG, the ontogeny of its biosynthesis and glycoform composition have not been established in the ovine fetus. Therefore we examined whether changes in plasma corticosteroid binding capacity (CBC) in fetal sheep during late gestation were associated with different patterns of glycosylation and reflected changes in tissue CBG expression. Since free cortisol is considered the bioactive fraction, we measured changes in the percent and absolute free cortisol in fetal plasma during late gestation. In order to examine whether CBG alters cortisol negative feedback at the level of the fetal pituitary, we also examined the effect of exogenous CBG in mediating the glucocorticoid-induced suppression of basal and corticotrophin-releasing hormone (CRH)-stimulated ACTH release from fetal pituitary cells in culture. The mean free cortisol concentration in plasma was not different between days 15 and 20 prior to parturition, and between 5 and 10 days prepartum, although it did rise between these times. Plasma CBC in chronically catheterized fetuses rose from 23.3 +/- 4.6 ng/ml at day 115 to 86.5 +/- 20.8 ng/ml at term and then decreased rapidly after birth. Between day 125 and day 140 of pregnancy approximately 10% of fetal plasma CBG was retarded by Concanavalin-A chromatography. This proportion increased at birth and attained adult values of > 70% by one month of age. By Northern blotting the relative levels of CBG mRNA in the fetal liver did not change between days 100 and 125, then increased significantly at day 140, but declined at term and in newborn lambs. CBG mRNA was undetectable in total RNA from lung, kidney, hypothalamus and placentomes, but was present in the fetal pituitary at days 125 and 140. Reverse transcription-PCR was used to confirm the presence of CBG mRNA in pituitary tissue from term fetuses. In cultures of term fetal pituitary cells, added CBG attenuated the cortisol- but not the dexamethasone-mediated suppression of basal and CRH-stimulated ACTH release. We conclude that in fetal sheep there is an increase in the corticosteroid binding capacity of plasma during late pregnancy which regulates, in part, free cortisol levels in the circulation. The liver is the major site of CBG biosynthesis in the fetus and at least until day 140 of gestation the rise in plasma CBC is associated with an increase in hepatic CBG mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of sex hormone-binding globulin mRNA and attenuation of corticosteroid-binding globulin mRNA by triiodothyronine in human hepatoma cells.

We examined the time course and dose response of the triiodothyronine (T3) effect on mRNAs for sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) in cells of the human hepatoma line HepG2. After 7 h of exposure to a saturating dose of T3, SHBG mRNA was unchanged but increased to 1.5 +/- 0.1 times the unstimulated control at 22 h. Maximal stimulation (2.3 +/- 0.6) was observed at 2-3 days. Corticosteroid-binding globulin mRNA was unchanged for 22 h after exposure to T3 but diminished thereafter to 64% by day 3. At 3-4 days of exposure, the changes in both SHBG mRNA and CBG mRNA were dose-responsive to the T3 concentration. For both mRNAs, half-maximal response occurred between 10 and 20 pmol/l bioavailable T3. Cortisol-binding proteins secreted by HepG2 cells after 3 days in culture also were T3 dose-responsive. No re-uptake of secreted CBG by the cells was observed, suggesting that the T3 effect on CBG secretion occurs during production of the mature protein. These data suggest that T3 stimulates the expression of the SHBG gene and attenuates the expression of the CBG gene. The effects of T3 on these genes are consistent with the increase in circulating SHBG and decrease in circulating CBG observed in hyperthyroidism. The HepG2 cells may be a useful human cell line in which to study the diversity of the molecular mechanisms of T3 action.