Roel Docter

Characteristics of active transport of thyroid hormone into rat hepatocytes

Thyroid hormone uptake into primary cultured rat hepatocytes was studied using 1-min incubations with radio-iodine-labelled iodothyronines. (1) Uptake of thyroxine indicates two saturable sites with apparent Km values of 1.2 nM and 1.0 microM, and non-saturable uptake. Similar kinetics of triiodothyronine uptake have been observed. (2) The high-affinity systems of both hormones are energy-dependent (i.e., inhibited by KCN and oligomycin). It is postulated that these systems represent active transport of thyroid hormone into the cell. (3) Analysis of mutual inhibition by the substrates for the triiodothyronine and thyroxine transport systems indicates that triiodothyronine and thyroxine cross the cell membrane via separate transport systems. (4) Preincubation with ouabain resulted in a decrease in uptake of both triiodothyronine and thyroxine, suggesting that a sodium gradient is essential for this transport.

Decreased transport of thyroxine (T4), 3,3',5-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) into rat hepatocytes in primary culture due to a decrease of cellular ATP content and various drugs.

T4, the main secretory product of the thyroid gland, is deiodinated and conjugated in peripheral tissues [ 1,2]. Phenolic ring deiodination of T4 accounts for -80% of the total body production of Ts, the most biologically active iodothyronine [3]. The remainder is produced by the thyroid gland. The other main product of peripheral deiodination is rT3, which is biologically inactive [4]. Initiation of biological activity by Ts occurs after binding to nuclear receptors [5]. At least 70% of the liver nuclear bound Ts is derived from the extracellular compartment and the remainder from local, intracellular deiodination of T4 [6]. The liver, which contains -30% of the total T4 pool and 80% of all intracellularly located T4, is an important organ for the production of thyroid hormone metabolites [7]. Studies related to membranal transport of iodothyronines into hepatic cells are important since they may increase our understanding of regulatory mechanisms involved in the ultimate delivery of thyroid hormone to intracellular active sites like metabolizing enzymes and receptors. fluorescent T3 in cultured iibroblasts [14,15]. We report here on a difference in ATPdependency of the uptake of T3 on the one hand and that of T4 and rTa on the other by rat hepatocytes in primary culture. T4 and rTa showed the most remarkable diminution of transport by small decreases in cellular ATP content. In addition, effects of propranolol, X-ray contrast agents, amiodarone and cytoskeleton-disrupting agents have been studied. The results indicate that besides changes in T4 deiodination [ 1,2] and sulfoconjugation [ 16,171, decrease in cellular uptake of T4 by tissues secondary to decreased cellular ATP concentrations or to the effects of some compounds may be a contributing factor to the clinical condition known as low T3 syndrome. A preliminary account of this work has been published [ 181.

Treatment of metastatic breast cancer patients with different dosages of megestrol acetate; dose relations, metabolic and endocrine effects.

Megestrol acetate (MA) is of therapeutic value in breast cancer patients. This study was designed to evaluate the effects of different dosages of MA on endocrine events potentially influenced by the drug in relation to plasma level of MA and clinical effects in patients with advanced breast cancer. Eighteen postmenopausal patients were randomly distributed over six groups to receive daily 90, 180 or 270 mg of MA (niagestin) orally in a cross-over study consisting of 3 periods of 6 weeks. Complete remission was observed in 1 patient, partial remission in 9, no change in 4 and failure in 4 patients. During the 18 weeks of treatment plasma levels of MA gradually increased, irrespective of the dose administered. Significant rises of the basal and TRH-stimulated plasma PRL and basal insulin levels were observed, whereas LH and FSH, estradiol, SHBG and the pituitary-adrenal axis were suppressed. None of these metabolic effects showed a correlation with the clinical response. We concluded that treatment of metastatic breast cancer with 180 mg MA/day is effective and causes minimal adverse effects.

Thyroxine Plus Low-Dose, Slow-Release Triiodothyronine Replacement in Hypothyroidism: Proof of Principle

Studies in hypothyroid rats show that, when infused with a combination of thyroxine (T4) plus triiodothyronine (T3) to normalize thyrotropin (TSH), euthyroidism in all organs is only ensured when T(4) and T(3) are administered in a ratio as normally secreted by the rat thyroid. As substitution with T(4)-only results in an abnormal serum T(4)/T(3) ratio, it is also possible that in humans, euthyroidism does not exist at the tissue level in many organs, considering that iodothyronine metabolism in the human and the rat share many similar mechanisms. Recent reports in which cognitive function and well-being are compared in patients with primary hypothyroidism substituted with T(4)-only versus substitution with T(4) plus T(3) result in controversial findings in that either positive or no effects were found. In all these studies T(3) was used in the plain form that results in nonphysiologic serum T(3) peaks. In these studies it is suggested that substitution with T(3 )should preferably be performed with a preparation that slowly releases T(3) to avoid these peaks. In the study reported here we show that treatment of hypothyroid subjects with a combination of T(4) plus slow-release T(3) leads to a considerable improvement of serum T(4) and T(3) values, the T(4)/T(3) ratio and serum TSH as compared to treatment with T(4)- only. Serum T(3) administration with slow-release T(3) did not show serum peaks, in contrast to plain T(3).

Inhibition of iodothyronine 5'-deiodinase by thioureylenes; structure--activity relationship.

goitrogenic activity [I]. These substances block the biosynthesis of thyroxine (T,) by inhibiting thyroid peroxidase [Z]. Among them rare the 2-bhiouracil (TU) derivatives, which have an additional inhibitory zffect on the deiodinactive metabolism of tky)iroid hormone in peripheral tissues [3,4]. T4 is considered as a prohormone, which is converted into the biologically active form of thyroid hormone, 3,3’,5triiodothyronine (T,), by the enzyme iodothyronine 5’deiodinase [5]. Deiodination of Tq may also yield the inactive rnetabolite 3,3’.5’-triiodothyronine (rT3). This latter reaction is probably mediated by a second enzyme, iodothyronine 5-deiodinase 151. The main pathway of rT; degradation is by 5’-deiodination into 3,3’-diiodothyronine [511. The latter may also be poduced by 5-deiodination of T, 151:

Anti-tumor and endocrine effects of chronic LHRH agonist treatment (Buserelin) with or without tamoxifen in premenopausal metastatic breast cancer.

SummarySeventeen premenopausal women with metastatic breast cancer were treated with the potent Luteinizing Hormone Releasing Hormone (LHRH) agonist Buserelin as a first-line agent. Twelve patients (group A) were treated with Buserelin alone and five patients (group B) with the combination of Buserelin and tamoxifen from the start of treatment. In nine patients of group A tamoxifen was added to Buserelin later on because of tumor progression or recurrent peaks of plasma estradiol (E2). Chronic intranasal therapy with Buserelin alone, preceeded by parenteral administration, caused an objective remission in four patients (2 × C.R., 2 × P.R.) and stable disease in four further patients without causing side effects. The longest duration of response until now is more than 29 months. After addition of tamoxifen a partial response occurred in two more patients of group A. Anovulation with suppressed progesterone secretion was reached in all patients treated with Buserelin alone, but transient peaks of E2 occurred in the majority (60%) of the patients. Addition of tamoxifen to Buserelin treatment caused disappearance of E2 peaks in 2 patients, but also reappearance of progesterone secretion with recurring E2 peaks in 3 other patients; in one case hyperstimulation of the ovaries was observed without progression of tumor growth. In group B only one woman showed a complete castration effect, while in four patients progesterone secretion was not (completely) suppressed. In two of these five patients an objective response occurred. In conclusion, Buserelin appears effective in the treatment of premenopausal women with metastatic breast carcinoma, but with the regimen used close control of endocrine parameters is necessary because of the variation in hormonal response with a risk of (hyper)stimulation of the ovaries, especially during combination therapy with tamoxifen.

Transport of thyroxine into cultured hepatocytes: effects of mild non-thyroidal illness and calorie restriction in obese subjects

OBJECTIVE Inhibitors of cellular T4 transport leading to diminished plasma T3 production have been identified as 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid (CMPF) and indoxyl sulphate in uraemia and bilirubin and non‐esterified fatty acids (NEFA) in critically ill patients with hyperbilirubinaemia. We question whether other factors are responsible for the altered thyroid hormone parameters observed in mild illness and during calorie restriction.

REGULATION OF THE ACTIVE TRANSPORT OF 3,3',5-TRIIODOTHYRONINE (T3) INTO PRIMARY CULTURED RAT HEPATOCYTES BY ATP

Translocation of thyroid hormone over the plasma membrane of hepatocytes and other cells is an essential step for intracellular deiodination [l] and binding to nuclear receptors [2] and other intracellular sites [3,4]. The nucleus of the target cell is supposed to be the site of initiation of thyroid hormone action [5,6]. Recent studies show that thyroid hormone binds to isolated plasma membranes of hepatocytes [7,8] and is actively transported into hepatocytes by means of a carrier mediated process [9-l 11. Evidence has been presented that T3 and thyroxine (T4) are translocated into the cells by different high-affinity, energy dependent mechanisms which can be blocked by ouabain [ 1 I]. This suggests that a sodium gradient over the cell membrane is essential for transport. In addition, T3 and T4 bind with low affinity to the plasma membrane at different sites [ 10,12]. Studies with human erythrocytes [13] showed similar kinetics of T3 transport and ouabain sensitivity as our previously published observations with hepatocytes [IO,1 11. We reported that (1) pre-exposure of hepatocytes in monolayer to increasing amounts of T3 results in a progressive decrease in the active transport of Ts into the cell. The extent of this diminution is dependent on time and hormone concentration and not on de nova protein synthesis, as cycloheximide does not interfere with this phenomenon. (2) Preexposure of the cells to T3 or fructose effected a decrease in total cellular ATP content. The positive correlation between the transport of T3 and total cellular ATP content suggests a causative relationship. We postulate that uptake in vivo of thyroid hormone by target cells is dependent on intracellular ATP levels. In

Adaptive changes in transmembrane transport and metabolism of triiodothyronine in perfused livers of fed and fasted hypothyroid and hyperthyroid rats

The transport and subsequent metabolism of triiodothyronine (T3) were studied in isolated perfused livers of euthyroid, hypothyroid, and hyperthyroid rats, both fed and 48-hour-fasted. T3 kinetics (transport and metabolism) during perfusion were evaluated by a two-pool model, whereas the metabolism of T3 was also investigated by determination of T3 breakdown products by chromatography of medium and bile. For comparison of groups, metabolism was corrected for differences in transport. Transport parameters in fed hypothyroid livers were not significantly changed as compared with euthyroid livers, whereas metabolism was decreased. In fed hyperthyroid livers, fractional transfer rate constants for influx (k21) and efflux (k12) were decreased and metabolism, corrected for differences in intracellular mass transfer, was increased. Furthermore, for transport in hyperthyroid liver it was shown that only total mass transfer (TMT) into the metabolizing liver compartment (not into the nonmetabolizing liver compartment) was decreased. Transport and metabolic parameters in fasted hypothyroid livers were decreased as compared with euthyroid fed livers. In fasted hyperthyroid livers, transport and metabolism were not significantly different as compared with that in euthyroid fed livers, so transport was increased versus hyperthyroid fed livers. It appeared therefore that fasting normalized the effects of hyperthyroidism on both the transport and metabolic processes of T3 in the liver. The present study demonstrates normal transport and decreased metabolism in livers of hypothyroid fed rats and decreased transport and increased metabolism in livers of hyperthyroid fed rats. In livers of hypothyroid fasted rats transport and metabolism were decreased, whereas in livers of hyperthyroid fasted rats transport and metabolism were not significantly different from that in euthyroid fed livers.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced T3 deiodination by the human hepatoblastoma cell line HepG2 caused by deficient T3 sulfation

Type I deiodination of T3 sulfate occurs at a Vmax that is 30-fold higher as compared to T3, both in rat and in human liver homogenates. We now present data showing lack of T3 deiodination by a human liver derived hepatoblastoma cell line, HepG2, caused by deficient T3 sulfation. Cellular entry of T3 was assessed by its nuclear binding after whole cell incubation. In spite of the presence of type I deiodinase, as confirmed by T4 and rT3 deiodination in homogenates, no deiodination of T3 could be detected. Since HepG2 cell homogenates also deiodinated chemically synthesized T3 sulfate (T3S) and inhibition of type I deiodination by propylthiouracil (PTU) did not cause T3S accumulation in whole cell incubations, we conclude that (i) HepG2 cells show reduced T3 deiodination caused by deficient T3 sulfation, and (ii) sulfation of T3 is an obligatory step prior to hepatic deiodination.