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An interval timing mechanism in the brain governs reproduction in seasonally breeding mammals by triggering refractoriness to inhibitory short photoperiods during midwinter. The neural mechanisms responsible for the timing and induction of photorefractoriness by this seasonal clock are unknown. Using cDNA microarrays and RT-PCR, we identified a class of genes encoding thyroxine (T4)-binding proteins (transthyretin, T4-binding globulin, albumin) whose expression is associated with reproductive refractoriness to short day lengths. Down-regulation of these genes was associated with reduced hypothalamic T4 uptake, which was reversed by long-day photoperiod treatments that restored responsiveness to short days. Circulating T4 concentrations did not vary with states of photoresponsiveness in euthyroid hamsters, but blockade of thyroid function accelerated the onset of photorefractoriness to short days. These data link changes in gene expression in the hypothalamus to the functional output of a seasonal clock. Reproductive inhibition in short days depends on T4 only late in the nonbreeding season. Down-regulation of genes encoding T4-binding proteins in the hypothalamus during this interval may restrict access of a static T4 signal to hypothalamic target tissues that regulate reproduction, thereby timing annual transitions in reproductive function. Hypothalamic autoregulation of T4 influx may constitute a critical cellular process involved in the generation and expression of seasonal reproductive rhythms and suggests a previously undescribed mechanism by which neural targets gain access to peripheral hormones.

The content of thyroxin-binding globulin isoforms differing by the carbohydrate constituent is significantly increased in the blood of coronary patients and patients with insulin-dependent diabetes mellitus compared to normal and is comparable to that in cancer patients. Enhanced biosynthesis of abnormal proteins due to structural and functional reorganization of plasma membranes is probably responsible for accumulation of thyroxin-binding globulin isoforms.

Thyroxine-binding globulin (TBG) is the main transport protein for thyroxine (T4) in blood. It shares considerable sequence homology with alpha 1-antitrypsin (AT) and other members of the serine proteinase inhibitor (serpin) superfamily of proteins. The crystallographic structure of AT has been determined and was found to represent the archetype of the serpins. This model has been used for structure-function correlations of TBG. Sequence analysis of the heat-resistant variant TBG-Chicago (TBG-CH) revealed a substitution of the normal tyrosine 309 with phenylalanine. For further analysis, vectors containing the coding regions of normal TBG (TBG-N) and TBG-CH were constructed, transcribed in vitro, and expressed in Xenopus oocytes. Both TBGs were secreted into the culture medium and could not be distinguished by gel electrophoresis. Scatchard analysis of T4 binding to TBG-N and -CH revealed no significant differences in binding affinity. The rate of heat denaturation of TBGs was determined by measurement of residual T4 binding capacity after incubation at 60 degrees C for various periods of time. The half-life values of denaturation of TBG-N and -CH were 7 and 132 min, respectively. The tyrosine 309 to phenylalanine substitution of TBG-CH involves a highly conserved phenylalanine residue of the serpins. The respective phenylalanine 312 of AT ties the alpha-helix hI1 to the molecule, thus stabilizing the tertiary structure. A substitution with tyrosine would disrupt this interaction. Accordingly, stabilization of the TBG molecule by replacement of tyrosine with phenylalanine in position 309 causes the increased heat stability of TBG-CH.

A sensitive [125I]-T4 binding assay was used to measure serum T4-binding globulin (TBG) in 60 individuals selected on the basis of their total circulating T3 concentrations, and a relationship between TBG and circulating thyroid hormone levels in humans was confirmed. There was a significant correlation between serum TBG and T3 or free T4 index. TBG secretion and TBG messenger ribonucleic acid (mRNA) production were studied with a continuous culture of the human hepatoblastoma cell line, HepG2. Cells were maintained in serum-free media for experimental manipulations. The addition of 100 nmol/L T3 to the cell medium resulted in a time-dependent down-regulation of TBG mRNA to 33 +/- 6% (+/- SD, n = 4) of untreated control levels by 24 h. Suppression of TBG mRNA was first detectable at 8 h (57% of untreated control levels). The effect of T3 was dose-responsive, with half-maximal suppression of TBG mRNA occurring at a bioavailable T3 concentration of approximately 30 pmol/L. The effect of T3 on TBG mRNA was not caused by a change in mRNA stability. Proteins secreted by HepG2 cells bound T4 with an affinity identical to that of normal circulating TBG. Cell secretion of TBG was parallel to total protein secretion and consistent with a TBG secretion rate of 50 ng/10(6) cells per day. Variations in the concentration of secreted binding protein in the presence of T3 corresponded to the changes observed in TBG mRNA. These data show that circulating TBG concentration is negatively correlated with total serum T3 in vivo. The corresponding down-regulation observed between TBG mRNA and secreted protein in HepG2 cells suggests that this effect is the result of the action of T3 on cellular TBG mRNA synthesis.

We have investigated the role of the thyroid compared with the hypophysis in the regulation of the two saturable thyroid hormone carriers of rat serum, thyroxine-binding globulin (TBG) and transthyretin (TTR). We examined, at serum and hepatic mRNA level, the responses of TBG and TTR to thyroidectomy (Tx), hypophysectomy (Hx) and replacement treatments with tri-iodothyronine (T3) or/and GH, both hormones which are depleted when the thyroid or hypophysis are removed. The studies were performed on male rats at the age of 8 weeks, when the developmentally regulated TBG becomes undetectable after its transient postnatal rise, while the nondevelopmentally regulated TTR presents its normal, age-independent level of expression. Tx-induced TBG re-expression was completely reversed by T3 replacement and unresponsive to GH replacement. TTR in the serum, on the other hand, was not affected by Tx or T3 replacement, moderately reduced by Tx in terms of the amount of mRNA, and markedly reduced by GH replacement. GH treatment, moreover, inhibited the expression of TTR in euthyroid controls. Hx, like Tx, induced TBG re-expression, an effect efficiently antagonized by T3 replacement. However, TBG synthesis was higher in Hx than in Tx rats and less effectively antagonized by T3 replacement. Most unexpectedly, GH induced a dramatic further increase in TBG synthesis, and the TBG synthesized in the GH-replaced Hx rats was entirely resistant to down-regulation by T3 replacement. TTR was markedly decreased at both serum and hepatic levels by Hx, unaffected by T3 and further decreased by GH replacement.(ABSTRACT TRUNCATED AT 250 WORDS)

A full-length thyroxine-binding globulin clone was isolated from a sheep liver cDNA library. The nucleotide sequence consisted of 1379 nucleotides and contained the coding region of 1236 nucleotides. The deduced sequence of the mature protein was 396 amino acids long. The similarity of the sheep thyroxine-binding globulin amino acid sequence with those of human and rat were 80% and 76%, respectively. Expression of the thyroxine-binding globulin gene was investigated by Northern analysis. The gene was not expressed in choroid plexus nor at other extrahepatic sites. The expression of the thyroxine-binding globulin gene was also studied during sheep foetal development. Thyroxine-binding globulin mRNA was already detected in livers of 1 cm long foetuses and levels similar to those found in liver from adult sheep were reached half way through pregnancy.

T4-binding globulin (TBG) is a glycoprotein of hepatic origin which transports thyroid hormone in serum. To characterize the human TBG (hTBG) gene, we studied its genomic organization, promoter activity, and regulation. To this purpose, we isolated from liver a complete hTBG cDNA clone containing the 5'-untranslated region and localized the transcription start site (TSS). The analysis of genomic clones revealed that the hTBG gene consists of five exons and that its exon-intron organization is similar to that of other members of the serine protease inhibitor family. The first exon (exon 0) is a short noncoding sequence located 1.62 kilobase pairs (kbp) upstream from exon 1. Potential cis-acting transcriptional regulatory elements including a TATA box, a CAAT box, and a hepatocyte nuclear factor-1 binding motif were identified in the upstream region. A reporter gene in which 3.2 kbp of the 5'-flanking region, including exon 0, was inserted upstream of the bacterial chloramphenicol acetyltransferase gene showed significant activity when transfected into a hepatblastoma-derived (HepG2) cell line. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, down-regulated the promoter activity by more than 80% and completely inhibited hTBG synthesis, whereas thyroid hormone, glucocorticoid, estrogen, and nicotinic acid had little, if any, effect. A series of 5'-deletions revealed that the fragment -218 to +4 from the TSS had the highest promoter activity, nearly 1000-fold greater than the promoterless chloramphenicol acetyltransferase construct. When nonhepatocyte-derived cell lines (CV-1 and CHO) were tested, promoter activity was reduced by a factor of 100, showing that the promoter works in liver-specific manner. The region -218 to -102 contains liver-specific enhancer elements, since deletion to nucleotide -101 resulted in a profound reduction of the promoter activity in HepG2 cells but not in CV-1 or CHO cells. On the other hand, mutational disruption of the putative hepatocyte nuclear factor-1 site (located 65 bp upstream of the TSS) completely abolished the promoter activity in all cell lines, indicating that this site is absolutely required for the transcription of the hTBG gene.

Thyroxine-binding globulin (TBG) is a glycoprotein that transports thyroid hormones in blood. Of two naturally occurring variants in man that harbor single proline substitutions (TBG-CD5 and TBG-Montreal), only TBG-CD5 manifests as complete TBG deficiency. In order to determine the pathophysiology of these TBG disorders, we expressed TBG-CD5 and TBG-Montreal (TBG-M), as well as the common type TBG (TBG-C) in reticulocyte lysate and Xenopus oocytes. Vectors encoding the three TBG types were constructed, transcribed in vitro, and their products of cell-free translation and processing by canine microsomal membranes were analyzed. TBG-C and TBG-M had identical mobility on denaturing polyacrylamide gel electrophoresis but could be distinguished by differences in thyroxine (T4) binding. TBG-CD5 had altered electrophoretic mobility and did not bind T4. TBG-C and TBG-M expressed in microinjected Xenopus oocytes showed properties similar to their respective serum forms, whereas TBG-CD5 was found in small amounts only intracellularly. Our results confirm that the previously described alanine 113 to proline substitution is responsible for the altered properties of TBG-M. The substitution of leucine 227 by proline in TBG-CD5 appears to impair its cotranslational processing and secretion.

T4-binding globulin (TBG) shares a high degree of homology with two serpin antiproteases, alpha 1-antichymotrypsin (ACT) and alpha 1-antitrypsin (AT), whose synthesis is increased during the acute phase phenomenon, which accompanies trauma, infections, and neoplasms. Interleukin-6 (IL-6) is believed to be the main effector of the acute phase response. When evaluated in human hepatoblastoma-derived (Hep G2) cells exposed to different doses of the recombinant human cytokine for variable time intervals, IL-6 caused a dose- and time-dependent decrease in the secretion of [35S]methionine-labeled TBG, transthyretin (TTR), and albumin. The secretion of ACT and AT was increased. These changes were not due to alterations in the secretory process, since the kinetics of secretion of newly synthesized proteins were not modified. IL-6 did, however, cause a decrease in the steady state levels of mRNA for TTR, TBG, and albumin and an increase in ACT and AT mRNAs. In addition, nuclear run-off assay demonstrated a decrease in the transcription of TTR, TBG, and albumin genes and an increased transcription of the ACT gene. Quantitation of the results showed that changes in the secretion of proteins, in steady state mRNA levels, and in gene transcription were superimposable for each protein, indicating that IL-6 exerts its effect on thyroid hormone-binding proteins mostly at the transcriptional level and that TTR is the thyroid hormone-binding protein showing the most pronounced negative regulation by IL-6. The opposite effect of IL-6 on TBG and the antiproteases, despite their structural homology, underscores gene divergence among these proteins.

Polyclonal (rabbit) antisera were generated against a high-affinity plasma thyroxine (T4) binding protein (TBP) purified from the turtle, Trachemys scripta, and used to develop a specific radioimmunoassay (RIA). The RIA demonstrated the presence of an immunochemically related protein in the plasma of several other species of Trachemys and in members of several other genera from the same family, Emydidae. Plasma from all nonemydids and some emydid genera either showed no competition or nonparallelism in RIA. The presence and level of radioimmunoassayable TBP in diverse species correlated with results of previous comparative measurements of T4 binding activity. However, an immunoreactive protein of the same molecular weight as TBP was identified in all turtles by Western blot analysis. More detailed studies in T. scripta demonstrated that variations in plasma T4 binding activity induced by experimental or environmental manipulations were related to differences in TBP concentrations. The concentration of TBP varied by orders of magnitude (from less than 1 to ca. 150 mg/liter) in euthyroid animals; levels showed ontogenetic changes (virtually absent in hatchlings) and were directly related to thyroidal status. Experimentally induced hypothyroidism (goitrogen treatment or surgical thyroidectomy) resulted in a marked suppression of TBP, and T4 treatment prevented its decline or reinstated it. Thus, in the turtle, this T4 transport protein may exist in higher concentrations and its levels are more variable and show a different relationship to thyroid activity than the analogous T4 binding globulin (TBG) in mammals.

The addition of sodium butyrate, a differentiation-inducing agent, to the culture medium of human hepatoblastoma-derived (Hep G2) cells, produced a dose-dependent and time-dependent increase in albumin (ALB) and decrease in T4-binding globulin (TBG) synthesis and secretion. In the presence of 0.01 to 2.0 mM sodium butyrate, newly synthesized [35S]ALB progressively increased up to 139% of control cultures grown in the absence of sodium butyrate, whereas TBG synthesis was already slightly inhibited using the lowest concentrations of this agent and further diminished thereafter. The use of 5 mM and 10 mM sodium butyrate inhibited the synthesis of both proteins, probably as a consequence of toxic effects on cell cultures. The addition of 1 mM sodium butyrate for variable time intervals caused an increase in the amount of ALB recovered in the medium up to 146% after 72 h, and a decrease of TBG up to 44% of controls. These different effects on ALB and TBG occurred concomitantly with an inhibition of cell growth, as shown by the reduction in the cell number/flask compared to control cultures. At the highest sodium butyrate concentrations, a relevant impairment in the secretion of newly synthesized TBG, but not of ALB, also occurred. These divergent effects on ALB and TBG synthesis by Hep G2 cells might be related to biochemical differentiation induced by sodium butyrate in this tumoral cell system, suggesting that TBG synthesis is increased in Hep G2 cells because of their neoplastic nature.

Using a human thyroxine binding globulin (TBG) cDNA probe, we demonstrate that rat liver contains two TBG mRNA species of different length, consisting of about 1.8 Kb and 2.4 Kb respectively. Slot blot analysis of the hepatic mRNAs from rats of different age reveals a fair correlation between the developmental trend of the messengers and that of the TBG circulating levels. Finally Northern blot and slot studies demonstrate that the increase of serum TBG induced in adults by thyroidectomy actually reflects an enhanced hepatic biosynthesis of the protein.

Acute psychiatric illness may be accompanied by transient hyperthyroxinemia. The mechanism of this phenomenon was examined by determining the role of thyrotropin (TSH) in the genesis of this state. Serial measurements of TSH, thyroxine (T4), free T4 index (FT4I), triiodothyronine (T3), and free T3 index (FT3I) were performed in 45 acutely hospitalized patients with major psychiatric disorders. Twenty-two (49%) patients exhibited significant elevations (greater than or equal to 2 SD above mean value of controls) of one or more thyroid hormone (or index) levels. Among depressed patients with elevated FT4I, TSH was higher (p less than .05) on the day of the peak FT4I than on the day of the FT4I nadir. There were significant positive correlations between psychiatric symptom severity and levels of FT4I among both depressed (p less than .01) and schizophrenic (p less than .025) patients. These data show that elevations of T4, FT4I, T3, and FT3I are common among psychiatric inpatients, especially early in their hospitalization, and that levels of thyroid hormones are correlated with severity of psychiatric symptomatology. TSH is higher early in the acute phase of illness and is not suppressed in the face of elevated thyroid hormone levels, a finding that distinguishes this phenomenon from ordinary hyperthyroidism. Elevations of peripheral thyroid hormone levels, particularly among depressed patients, may result from a centrally-mediated hypersecretion of TSH.

Human chorionic gonadotrophin (hCG) shares structural similarity with pituitary thyrotrophin (TSH) and may act as a thyroid stimulator. We have studied serum hCG levels, thyroid function tests and the ability of serum to stimulate cultured thyroid cells in 40 subjects between 6 and 12 weeks of pregnancy. Serum free tri-iodothyronine was increased and serum TSH reduced in pregnancy samples (both p less than 0.05). hCG was detectable in all pregnancy sera with a mean level of 105.6 X 10(3) U/l. Serum from 24 of the 40 (60%) patients stimulated iodide uptake into cultured FRTL-5 thyroid cells. The potency of sera in stimulating cells correlated with the hCG level (r = 0.710, p less than 0.01). The stimulatory activity in some, but not all, sera could be specifically neutralized with antiserum to hCG. Partially purified hCG stimulated iodide uptake and growth of thyroid cells at concentrations of 50 X 10(3) U/l and above. In these experiments, 25 X 10(3) U/l of hCG produced equivalent stimulation to 1 mU/l of TSH. In 8 patients tested before and after termination of pregnancy, the thyroid-cell-stimulatory activity of serum declined rapidly in parallel with serum hCG. hCG may stimulate the thyroid gland at concentrations which prevail in normal pregnancy. Its potential as a physiological regulator of the thyroid gland is not widely appreciated and requires further study.

Thyroxine-binding globulin (TBG) synthesis by human hepatoma (Hep G2) cells was demonstrated by pulse labeling with [35S]methionine or [3H]mannose and subsequent immunoprecipitation in the medium or cell lysate. Secreted TBG was glycosylated and had the same apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as TBG purified from human serum. Pulse-chase experiments failed to show any precursor form intracellularly. Treatment of cells with the amino acid analogs, canavanine and thialysine, did not cause secretion of large-molecular-weight moieties, in contrast to what was observed in the case of albumin. Thyroxine-binding activity, as assessed by [125I]thyroxine immunoprecipitation with anti-TBG serum, was detectable in the media of oocytes injected with RNA from Hep G2 cells. Translation of this RNA in rabbit reticulocyte lysate, followed by immunoprecipitation with anti-TBG serum, revealed a protein having the same electrophoretic mobility as deglycosylated TBG purified from human serum (Mr approximately 45,000). Since deglycosylated TBG still contains 3% of its weight as carbohydrate, it appears that the translation product contains an additional fragment (signal peptide) of about 1,500 daltons. It is unlikely, however, that TBG is synthesized via a larger-molecular-weight precursor.

It has been previously suggested that inherited thyroxine-binding globulin (TBG) abnormalities in man may be due to mutations at a single X-chromosome-linked locus controlling TBG synthesis. However, abnormalities in TBG degradation have not been excluded. The availability of purified human TBG and its successful labeling with radioiodide allowed us to examine such possibility. Human TBG was purified by affinity chromatography, labeled under sterile conditions with 131I or 125I,, and mixed with [125I]thyroxine (T4) or [131I]T4, respectively, before their intravenous injection. Blood and urine samples were collected over a 10-day period, and the turnover parameters were calculated. In eight normal volunteers mean values +/-SD for TBG and T4 respectively, were as follows: Half time (t1/2) 5.3 +/- 0.4 and 7.0 +/- 0.6 days; distribution space (DS) 7.2 +/- 1.0 and 10.8 +/- 1.2 liters; and total daily degradation (D) 0.211 +/- 0.053 and 0.088 +/- 0.011 mumol/day. In all subjects, t1/2 of TBG was shorter than that of T4; and the DS was smaller. 2.4 mol of TBG was degraded for each mole of T4. In five of six subjects from four families, comprising hemizygous and heterozygous carriers of TBG absence, decrease, and excess, the t1/2 and DS for TBG were within the normal range. The D of TBG was proportional to the serum concentration of the protein. Changes in the T4 kinetics in these patients were compatible with euthyroidism and with the known alterations in the extrathyroidal T4 pool associated with the changes in serum TBG concentration. A striking decrease in the t1/2 of TBG was found only in a patient with acquired diminution in TBG concentration and in patients with thyrotoxicosis or other conditions apparently unrelated to thyroid dysfunction. TBG t1/2 was 2.5 days in a patient with multiple myeloma and 3.6 days in two patients with thyrotoxicosis. Decreased TBG t1/2 was also observed in three of six patients with nonthyroidal pathology and was associated with an increase in TBG D disproportionate to their level of serum TBG. These studies indicate that changes in TBG concentration in patients with X-chromosome-linked TBG abnormalities are due to alterations in its rate of synthesis. In other conditions, abnormalities of TBG degradation and/or rate of synthesis may be found.

Thyroxine-binding globulin biosynthesis was demonstrated in hepatocytes isolated from normal adult Rhesus monkeys. Dispersed cells were obtained by in situ liver perfusion with collagenase, hyaluronidase and EDTA. Conditions for optimum cell survival and incorporation of radioactive leucine into newly synthesized proteins were defined. Protein synthesis, and specifically thyroxine-binding globulin synthesis, were shown to continue throughout the incubation period, while cell survival remained high (75% excluded trypan blue after 6h). Incubation medium, cytosol and a particulate fraction (extracted with digitonin) were analyzed for thyroxine-binding globulin. After extensive dialysis and purification by affinity chromatography, newly synthesized thyroxine-binding globulin was identified by specific double-antibody immunoprecipitation and by immunodiffusion and immunoelectrophoresis with autoradiography. Newly synthesized thyroxine-binding globulin was present after 4 h of incubation. After 6 h, the total synthesized had increased to 150% of the 4 h value, while the fraction present in the medium and increased to 300%, indicating probable thyroxine-binding globulin secretion

To investigate the effects of estrogen on thyroxine-binding globulin (TBG) metabolism, 4 female Rhesus monkeys were studied before and 3-4 weeks after implantation of beta-estradiol (E2)-containing capsules. In addition, 2 of the animals were also studied for the first 7 days after the start of E2. Serum E2 increased 10-fold from 20 +/- 7 to 212 +/- 41 pg/ml. Serum TBG, initially 20.2 +/- 6 mug/ml, was elevated by 24 h after E2 implantation, and reached a steady level of 46.8 +/- 5.0 mug/ml by 7-10 days. For the turnover studies, highly purified [125I]iodo-TBG was injected iv and serum [125I]PBI and urinary 125I excretion were measured daily. TBG kinetics were evaluated by use of a compartmental model. Although a 2-compartment model was sufficient to fit the control and late E2 data, a 3-compartment model was developed in order to account for the modifications observed during the early E2 period. The final decay rate (k) of TBG was 0.26 +/- 0.01/day during the control period and was slightly lower after E2 (0.23 +/- 0.01/day). In the 2 monkeys studied during the early E2 period, the major effect of E2 was a stimulation of the TBG production rate. This was simulated in the model by a stepwise increase occurring in the last quarter of the first day after E2. There was also an abrupt redistribution of TBG in the compartments defined by the model. The total distribution or serum equivalent volume of TBG after 3-4 weeks of E2 increased 1.4-fold, from 338 +/- 37 ml to 458 +/- 22 ml, and the metabolic clearance rate increased 1.3-fold, from 90 +/- 10 ml/d to 113 +/- 12 ml/d. The increase in the final TBG production rate (2.9-fold) was only slightly greater than the rate calculated for the early E2 period, and was similar to the increase we have recently found in monkey hepatocytes studied in vitro after isolation from E2-treated animals. It appears that stimulation of hepatic synthesis of TBG accounts for the elevated serum levels of TBG observed after estrogen.

The rate of in vitro production of thyroxine-binding globulin (TBG) was studied in hepatocytes isolated from 6 control rhesus monkeys (serum TBG: 19.6 +/- 0.5 micrograms/ml; mean +/- SE) and 6 monkeys treated for 4-5 weeks with beta-estradiol (E2) (serum TBG: 45.1 +/- 1.8 micrograms/ml). Incorporation of [3H]leucine into intracellular soluble and particle-bound TBG, and into secreted TBG was determined for incubation periods up to 9 h. TBG was purified by affinity chromatography and measured by specific immunoprecipitation. The absolute amount of [3H]TBG and the ratio of [3H]TBG to total labeled protein in the same fraction were 3-fold higher in the particulate fraction and in the incubation medium of hepatocytes isolated from E2-treated monkeys. In separate experiments, TBG accumulation in the medium was measured for periods up to 19 h by radioimmunoassay. A 2.4-fold increase was observed with hepatocytes from E2-treated monkeys (3.48 ng TBG/h/10(7) cells, compared to 1.46 in controls). Correction of the production rates for the number of cells surviving during the incubation, and assuming 10.2 x 10(9) cells per liver, gave TBG production rates of 250 micrograms/liver/day in hepatocytes from E2-treated monkeys and 104 micrograms/day in hepatocytes from control monkeys. These experiments demonstrate that estrogen increases in vitro synthesis and secretion of TBG by isolated hepatocytes. The observed 2.4 to 3-fold increase was similar to the 2.9-fold increase in TBG production measured in vivo by kinetic analysis of TBG metabolism.

The functional maturation of an independent foetal thyroid activity was investigated in the present study. Serum concentrations of total T4, free T4, TSH and TBG were measured in 23 foetuses between 18 and 31 weeks' gestational age. Foetal samples were collected by transabdominal needling from the placental cord insertion. TT4, FT4, TBG and TSH levels significantly increased with gestational age. FT4 levels were comparable with the adult range by 28 weeks' gestation; TBG levels reached adult values at approximately 30 weeks, while TT4 was lower than adult levels throughout the whole period studied. TSH values were, in all cases, higher than the normal adult range. A significant positive correlation was present between TT4 and TBG, TT4 and TSH, and TBG and TSH levels; on the contrary, no correlation was demonstrated between FT4 and TSH levels. The TSH/TT4 ratio significantly decreased with gestational age. The results suggest an incomplete responsiveness of the foetal thyroid gland to TSH, while the feedback control system between pituitary and thyroid is operating at a different set point from that in post-natal life. The normal range of thyroid parameters established is of clinical relevance for the antenatal diagnosis, and eventual treatment of thyroid disorders that may seriously damage foetal development and maturation.

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.

Serum thyroxine-binding globulin (TBG), the major plasma transport protein for thyroid hormones in man, was shown to be altered under the influence of estrogen and in hypothyroidism. In order to study these alterations, we used an animal model. Synthesis of TBG was demonstrated in hepatocytes isolated from adult Rhesus monkeys, and in a monkey hepatocarcinoma continuous cell culture line (NCLP-6-E). When the hepatocytes were obtained from monkeys pretreated with beta-estradiol (E2), a specific 2.5-2.9 fold increase of TBG synthesis and secretion was shown; similar data were obtained with the tumor line. Furthermore, an increased TBG production was shown with these cells when T4 (from 10(-14) to 10(-11) M) was added to the culture medium. The in vitro results were correlated with in vivo data obtained by investigating the metabolism of TBG after iv injection of tracer doses of purified, radiolabelled TBG to normal, E2-treated and thyroidectomized monkeys. The main effect of estrogen administration was a marked increase of the production rate. During hypothyroidism, the catabolism and the production rate of TBG were decreased. In both conditions, there were significant changes in the distribution volume of TBG, the physiologic relevance of which remains to be investigated.

Hyperestrogenemia in humans increases both the concentration of serum T4-binding globulin (TBG) by 2- to 3-fold and the proportion having anodal mobility on isoelectric focusing (IEF). As TBG is synthesized in the liver, we studied the effect of estrogen on TBG synthesis, secretion, and degradation by cultured human hepatocarcinoma cells (Hep G2). beta-Estradiol in concentrations in the range found in pregnancy (10(-7) M) had no effect on the accumulation of immunoreactive TBG in medium over 4 days. The absence of fetal calf serum or phenol red did not alter these findings. The amount of [35S]TBG accumulated 6 h after addition of [35S]methionine was not influenced by exposure to estrogen or to serum obtained from pregnant women. However, 10(-5) M beta-estradiol suppressed TBG more severely than albumin synthesis (34% vs. 9%). The lack of an estrogen effect on TBG synthesis and secretion was supported by experiments showing no effect of estrogen on the disappearance of TBG added to the medium or the accumulation of cytoplasmic TBG mRNA. The same cultures responded to estrogen by a 10-fold increase in nuclear estrogen receptor binding sites and a 2-fold increase in apolipoprotein CII. As TBG in serum, the rate of heat denaturation was not altered in TBG synthesized by Hep G2 cells in the presence of estrogen. In contrast to the effect on TBG in serum, in Hep G2 cells estrogen did not produce an anodal shift on IEF, or increased its proportion not bound to Concanavalin A, nor reduced its clearance rate when injected into rats. However, even untreated Hep G2 cells synthesized TBG with a larger number of anodal IEF bands and proportion of Concanavalin A excluded material than TBG in pregnancy serum. Results support our hypothesis, based on analysis of TBG in pregnancy, that estrogen-induced serum TBG elevation may not be mediated through an increase in synthesis. The failure to observe estrogen induced changes in oligosaccharide structure does not exclude estrogen responsivity of Hep G2 cells. Such effect could be masked by the marked constitutive increase in number of oligosaccharide chain antennae typical in this and other neoplastic tissues.

L-Asparaginase (ASNase), a drug widely used in the treatment of acute lymphoblastic leukemia, has been reported to decrease serum T4-binding globulin (TBG) levels, while results of serum albumin determinations were conflicting. This effect in vivo has been attributed to depressed liver protein synthesis, but this hypothesis has not been proved. To investigate this problem, human hepatoma (Hep G2) cells were continuously labeled for 4 h with 100 microCi/ml [35S]methionine in the absence or presence of graded amounts of ASNase (from 0.1 nM to 0.1 mM). Media and cell lysates were collected, immunoprecipitated with antialbumin or anti-TBG serum and protein A, and submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were sliced, and the radioactivity was counted in a beta-counter. A dose-dependent inhibition of TBG and albumin biosynthesis (as well as of total protein synthesis) was demonstrable, but TBG appeared to be more sensitive to the action of the drug. In fact, TBG biosynthesis was reduced by 8% with 0.1 nM ASNase, while an effect on albumin was observed only at 1 nM ASNase; 50% inhibition was obtained with 30 nM ASNase in the case of TBG and with 800 nM in the case of albumin. At the highest concentration (0.1 mM), TBG biosynthesis was reduced by 94%, and albumin biosynthesis by 75%. ASNase also proved to have a time-dependent effect, as assessed by the measurement of radioimmunoassayable TBG in the media from Hep G2 cells grown in the presence of 10 nM ASNase for 1-4 days. The TBG concentration was progressively reduced, by 40% after 1 day to 85% after 4 days. In pulse-chase experiments, a reduction of total (intracellular plus secreted) immunoprecipitable TBG and, to a lesser extent, albumin was observed, suggesting that the drug also affected the catabolism of newly synthesized proteins. These results provide the first in vitro evidence that ASNase actually inhibits TBG biosynthesis. This effect is not specific for TBG, but this protein appears to be more susceptible than albumin to ASNase action. This can explain why in patients treated with ASNase for leukemia, a decrease in serum TBG concentrations has not always been associated with a reduction in serum albumin levels.

In order to examine the action of thyroid hormone on the secretory proteins of the liver, we investigated the effects of thyroid hormones on the synthesis of T4-binding globulin (TBG), alpha 1-acid glycoprotein (AGP), and albumin in a human hepatoblastoma cell line, Hep G2. Hep G2 cells grown to be confluent in medium with 10% fetal calf serum were further cultured in serum-free medium for 4 days, and followed by treatment with hormones for 2 days changing the medium every 24 h. On day 2 (the second 24 h of hormone treatment), about 30% of TBG accumulation was inhibited by 10(-12) M T3 and 50% was inhibited by 10(-8) M T3, although no change was observed on day 1 (the first 24 h of hormone treatment). This inhibitory effect of T3 closely resembled the effect of T3 on [35S]methionine-labeled TBG synthesis by the cells incubated for 3 h after 42 h of pretreatment. About 30-55% of the newly synthesized [35S]TBG immunoprecipitated with anti-TBG serum was inhibited by 10(-8) M T3. These results showed that thyroid hormone inhibited TBG synthesis in Hep G2 cells. On the other hand, T3 stimulated the accumulation of AGP in the media on day 1 (140% of control by 10(-8) M T3), and the effect increased drastically on day 2 (250% of control by 10(-8) M T3). No effect of T3 on albumin accumulation or total protein synthesis was seen. The concentrations of T4 which had significant effects on TBG and AGP accumulation were 10 and 10(3) times higher than those of T3, respectively. In conclusion, thyroid hormone has dualistic effects on the secretory proteins synthesized by a human hepatoblastoma cell line: physiological concentrations of thyroid hormones decrease the synthesis of TBG, but increase the synthesis of AGP.

A two-step procedure to incorporate 125I-iodotyrosine into protein synthesized in a reticulocyte lysate is described. In the first step, the iodination of tyrosyl tRNA was catalyzed by a solid-state glycouril compound. More than one-third of 200 microCi of radioiodine became bound to 70 micrograms of aminoacyl tRNA after 15 min at 0 degrees C. The isotope was distributed in a three-to-one ratio of monoiodotyrosine to di-iodotyrosine. In the second step, the soluble product of the radioiodination was transferred directly into a nuclease-treated reticulocyte lysate coded with RNA isolated from the human hepatoma cell line Hep G2. Fractional recovery of radioiodine in nascent protein was maximally 7.6%. Reaction of the product of translation with antibody against alpha-antitrypsin separated an 125I-containing protein having a molecular weight estimated as 47,000. The synthesis of unprocessed alpha-antitrypsin was confirmed by cleavage of the labeled protein with leader peptidase and by its displacement from immunocomplex formation with purified alpha-antitrypsin. The amount of 125I incorporated into alpha-antitrypsin was proportionate to iodinated tRNA additions up to a concentration of 70 micrograms/ml. The synthesis of alpha-antitrypsin as detected in radioautograms after gel electrophoresis was more than twice as sensitive using radioiodinated aminoacyl tRNA as compared with [35S]methionine. Iodine labeling of thyroxine-binding globulin was also demonstrated in the translation product of Hep G2 RNA. Since the specific activity of the radioiodine is high and the means for detection of the isotope efficient, the method described can facilitate the demonstration of quantitatively minor translation products.

Human hepatoma (Hep G2) cells were shown to synthesize and secrete a novel T4-binding protein, called 27K protein for its apparent mol wt on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The mRNA coding for this protein was characterized by immunoprecipitation of [125I]T4 bound to 27K protein secreted into the medium of oocytes injected with total Hep G2 RNA. Sucrose gradient fractionation of RNA from Hep G2 cells showed that TBG mRNA and 27K mRNA had different sizes, indicating that TBG and 27K protein are two distinct proteins. In vitro translation of RNA in a rabbit reticulocyte lysate demonstrated that the translation product immunoprecipitated by anti-27K serum had the same mol wt as the immunoprecipitated protein from whole cells labeled with [35S]methionine, thus suggesting that 27K protein is neither derived from TBG nor synthesized through a larger mol wt precursor, and also that it does not contain carbohydrates. The absence of carbohydrates was further supported by the observation that [3H]mannose was not covalently bound to the 27K protein when Hep G2 cells were labeled with [3H]mannose, nor was there a shift in apparent mol wt when the cells were treated with the glycosylation inhibitor tunicamycin. The kinetics of secretion of 27K protein were similar to those of albumin and faster than those of TBG, which is also in keeping with the nonglycoprotein nature of 27K protein.

Five families with inherited thyroxine-binding globulin (TBG) abnormalities were studied. On the basis of serum thyroxine (T(4))- binding capacity of TBG in affected males, three family types were identified: TBG deficiency, low TBG, and high TBG capacity. In all families evidence for X-linked inheritance was obtained and in one family all criteria establishing this mode of inheritance were met. Only females were heterozygous, exhibiting values intermediate between affected males and normals. Overlap in heterozygotes was most commonly encountered in families with low TBG. QUANTITATIVE VARIATION IN THE SERUM CONCENTRATION OF FUNCTIONALLY NORMAL TBG WAS DEMONSTRATED BY: (a) failure of serum from TBG-deficient subjects to react with anti-TBG antibodies; (b) normal kinetics of T(4) and triiodothyronine-binding to TBG in sera from subjects with low TBG and high TBG capacity; (c) concordance of estimates of TBG concentration by T(4) saturation and by immunological methods; and (d) normal rate of heat inactivation of TBG. No abnormalities in serum transport of cortisol, testosterone, aldosterone, or thyroxine bound to prealbumin could be detected. These observations suggest that all the TBG abnormalities thus far observed reflect mutations at a single X-linked locus involved in the control of TBG synthesis.