Georg Hennemann

Thyroid Hormone Transport by the Heterodimeric Human System L Amino Acid Transporter

Transport of thyroid hormone across the cell membrane is required for thyroid hormone action and metabolism. We have investigated the possible transport of iodothyronines by the human system L amino acid transporter, a protein consisting of the human 4F2 heavy chain and the human LAT1 light chain. Xenopus oocytes were injected with the cRNAs coding for human 4F2 heavy chain and/or human LAT1 light chain, and after 2 d were incubated at 25 C with 0.01–10 M [ 125 I]T4, [ 125 I]T3 ,[ 125 I]rT3 ,o r [ 125 I]3,3-diiodothyronine or with 10 –100 M [ 3 H]arginine, [ 3 H]leucine, [ 3 H]phenylalanine, [ 3 H]tyrosine, or [ 3 H]tryptophan. Injection of human 4F2 heavy chain cRNA alone stimulated the uptake of leucine and arginine due to dimerization of human 4F2 heavy chain with an endogenous Xenopus light chain, but did not affect the uptake of other ligands. Injection of human LAT1 light chain cRNA alone did not stimulate the uptake of any ligand. Coinjection of cRNAs for human 4F2 heavy chain and human LAT1 light chain stimulated the uptake of phenylalanine > tyrosine > leucine > tryptophan (100 M) and of 3,3-diiodothyronine > rT3 T3 > T4 (10 nM), which in all cases was Na independent. Saturation analysis provided apparent Michaelis constant (Km) values of 7.9 M for T4, 0.8 M for T3, 12.5 M for rT3, 7.9 M for 3,3-diiodothyronine, 46 M for leucine, and 19 M for tryptophan. Uptake of leucine, tyrosine, and tryptophan (10 M) was inhibited by the different iodothyronines (10 M), in particular T3. Vice versa, uptake of 0.1 M T3 was almost completely blocked by coincubation with 100 M leucine, tryptophan, tyrosine, or phenylalanine. Our results demonstrate stereospecific Na-independent transport of iodothyronines by the human heterodimeric system L amino acid transporter. (Endocrinology 142: 4339 – 4348, 2001)

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.

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:

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

Inhibition of iodothyronine deiodinase by phenolphthalein dyes: structure--activity relationship.

It has been found that several iodine-containing radiographic contrast agents are potent inhibitors of the 5’-deiodination of thyroxine (T4) to 3,3’,5-triiodothyronine (T3) in peripheral tissues in vivo [ 1 ] as well as in vitro [2,3]. In rat liver microsomes the 5’-deiodination of 3,3’,5’-triiodothyronine (rT3) to 3,3’-diiodothyronine (3,3’-T2) and of 3’,5’-diiodothyronine to 3’-monoiodothyronine as well as the S-deiodination of T3 to 3,3’-TZ are also inhibited by these radiographic agents [4,5]. It is still uncertain whether a single enzyme mediates both types of deiodination or that two separate enzymes are involved, i.e., iodothyronine 5and 5’-deiodinase [5,6]. During attempts to purify the enzyme(s) from rat liver by electrophoresis, it was noted that the tracking dye bromophenol blue strongly inhibited deiodination. This compound is structurally related to iodothyronines and several X-ray contrast agents in that it also contains two halogen substituents in the ortho positions to an electron-donating group (OH or NH2). It was therefore thought of interest to study the structure-activity relationship of phenolphthalein derivatives as inhibitors of iodothyronine 5’-deiodination. Another point of consideration for this study was the wide use of these compounds as acid-base indicators. It was found that bromophenol blue is a very strong competitive inhibitor of iodothyronine 5’-deiodinase activity. At the same time sulfobromophthalein (BSP) and bilirubin, both compounds known to displace T4 and T3 from cytosol binding proteins [7], were found to inhibit the 5’-deiodination of rT3. 2. Materials and methods

THYROXINE BINDING GLOBULIN DEFICIENCY IN A FAMILY WITH TYPE I HYPERLIPOPROTEINAEMIA

A familial type I hyperlipoproteinaemia is described in three members of a family of eleven; on the basis of LPL activity and HDL content of plasma three other members of the family have been diagnosed to be heterozygotes without other disturbances in their lipid spectrum. The distribution of this lipid disorder is in accordance with an autosomal recessive inheritance pattern. In this family a second hereditary condition, thyroxine binding globulin deficiency, was found in addition to the hyperlipoproteinaemia. The inheritance of this condition appears to be as an autosomal dominant. An interrelated inheritance pattern of both conditions could not be proved, but both traits may be located on the same chromosome at some distance from another to allow recombination.

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.

EURO-COLLINS SOLUTION VERSUS UW-SOLUTION FOR LONG-TERM LIVER PRESERVATION IN THE ISOLATED RAT-LIVER PERFUSION MODEL

To compare UW-solution (UW) and Euro-Collins (EC) for long-term liver preservation we investigated the morphology and metabolic capacity of rat liver after 18 and 42-hours cold-storage in either UW or EC. After harvesting the rat liver was transferred to a perfusion chamber where it was perfused for 10 min with UW or EC at 4°C. Thereafter livers were stored at 4°C in UW or EC for 18 hours (both groups n = 6) or for 42 hours (both groups n = 8). After 18-hr or 42-hr cold-storage a 2-hr warm perfusion (37°C) was started with Krebs-Ringer solution with carbogen to which 125Iodine-triiodothyronine (T3) was added. Control livers (n = 8) were immediately perfused with Krebs-Ringer without cold-storage. The following parameters were assessed: ASAT-levels in the perfusate, T3-metabolites in the bile and the perfusate, the perfusion pressure, the volume of bile secreted and light-microscopical morphology at the end of the warm perfusion period. After cold storage in UW-solution the ASAT-levels in the perfusate were lower than after storage in EC as well as the perfusion pressures. These livers demonstrated a better T3-metabolism and secreted more bile than EC-stored livers. Histological examination showed more tissue damage in the EC-stored livers than in the UW stored livers. We conclude that cold-storage of rat liver in UW-solution resulted in a better morphology and metabolic capacity as compared with EC-solution.