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 livers 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. These changes might favor tissue euthyroidism despite the altered thyroid and nutritional state, and can therefore be seen as adaptation mechanisms to these altered states at the tissue level

Rapid sulfation of 3,3',5'-triiodothyronine in native Xenopus laevis oocytes

Sulfation is an important metabolic pathway facilitating the degradation of thyroid hormone by the type I iodothyronine deiodinase. Different human and rat tissues contain cytoplasmic sulfotransferases that show a substrate preference for 3,3'-diiodothyronine (3,3'-T2) > T3 > rT3 > T4. During investigation of the expression of plasma membrane transporters for thyroid hormone by injection of rat liver RNA in Xenopus laevis oocytes, we found uptake and metabolism of iodothyronines by native oocytes. Groups of 10 oocytes were incubated for 20 h at 18 C in 0.1 ml medium containing 500,000 cpm (1-5 nM) [125I]T4, [125I]T3, [125I]rT3, or [125I]3,3'-T2. In addition, cytosol prepared from oocytes was tested for iodothyronine sulfotransferase activity by incubation of 1 mg cytosolic protein/ml for 30 min at 21 C with 1 microM [125I]T4, [125I]T3, [125I]rT3, or [125I]3,3'-T2 and 50 microM 3'-phosphoadenosine-5'-phosphosulfate. Incubation media, oocyte extracts, and assay mixtures were analyzed by Sephadex LH-20 chromatography for production of conjugates and iodide. After 20-h incubation, the percentage of added radioactivity present as conjugates in the media and oocytes amounted to 0.9 +/- 0.2 and 1.0 +/- 0.1 for T4, less than 0.1 and less than 0.1 for T3, 32.5 +/- 0.4 and 29.3 +/- 0.2 for rT3, and 3.8 +/- 0.3 and 2.3 +/- 0.2 for 3,3'-T2, respectively (mean +/- SEM; n = 3). The conjugate produced from rT3 was identified as rT3 sulfate, as it was hydrolyzed by acid treatment. After injection of oocytes with copy RNA coding for rat type I iodothyronine deiodinase, we found an increase in iodide production from rT3 from 2.3% (water-injected oocytes) to 46.2% accompanied by a reciprocal decrease in rT3 sulfate accumulation from 53.7% to 7.1%. After 30-min incubation with cytosol and 3'-phosphoadenosine-5'-phosphosulfate, sulfate formation amounted to 1.8% for T4, less than 0.1% for T3, 77.9% for rT3, and 2.9% for 3,3'-T2. These results show that rT3 is rapidly metabolized in native oocytes by sulfation. The substrate preference of the sulfotransferase activity in oocytes is rT3 >> 3,3'-T2 > T4 > T3. The physiological significance of the high activity for rT3 sulfation in X. laevis oocytes remains to be established

Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability

Although it was originally believed that thyroid hormones enter target cells by passive diffusion, it is now clear that cellular uptake is effected by carrier-mediated processes. Two stereospecific binding sites for each T4 and T3 have been detected in c

Changes in renal tri-iodothyronine and thyroxine handling during fasting

OBJECTIVE: Liver handling of thyroid hormones (TH) has been known to alter significantly during fasting. This study investigates whether renal handling of TH is also changed during fasting. METHODS: We measured urinary excretion rates and clearances of free tri-iodothyronine (T(3)) and free thyroxine (T(4)) in healthy subjects prior to and on the third day of fasting. RESULTS: During fasting, both mean T(3) and T(4) urinary excretion decreased significantly to a mean value of 42% of control. Also, total and free (F) serum T(3) concentrations declined significantly, but serum T(4) did not change. Both FT(3) and FT(4) clearance decreased significantly during fasting (62% and 42% of control). The fasting-induced decrease in uric acid clearance correlated well with the decrease in FT(3) clearance (r=0.94; P<0.001). Serum concentrations of non-esterified fatty acids (NEFA) were significantly elevated during fasting. CONCLUSIONS: The findings cannot be fully explained by the fasting-induced decrease in serum T(3), a

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 microM [(125)I]T(4), [(125)I]T(3), [(125)I]rT(3), or [(125)I]3,3'-diiodothyronine or with 10-100 microM [(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 microM) and of 3,3'-diiodothyronine > rT(3) approximately T(3) > T(4) (10 nM), which in all cases was Na(+) independent. Saturation analysis provided apparent Michaelis constant (K(m)) values of 7.9 microM for T(4), 0.8 microM for T(3), 12.5 microM for rT(3), 7.9 microM for 3,3'-diiodothyronine, 46 microM for leucine, and 19 microM for tryptophan. Uptake of leucine, tyrosine, and tryptophan (10 microM) was inhibited by the different iodothyronines (10 microM), in particular T(3). Vice versa, uptake of 0.1 microM T(3) was almost completely blocked by coincubation with 100 microM leucine, tryptophan, tyrosine, or phenylalanine. Our results demonstrate stereospecific Na(+)-independent transport of iodothyronines by the human heterodimeric system L amino acid transporter

Expression of rat liver cell membrane transporters for thyroid hormone in Xenopus laevis oocytes

The present study was conducted to explore the possible use of Xenopus laevis oocytes for the expression cloning of cell membrane transporters for iodothyronines. Injection of stage V-VI X. laevis oocytes with 23 ng Wistar rat liver polyadenylated RNA (mRNA) resulted after 3-4 days in a highly significant increase in [125I]T3 (5 nM) uptake from 6.4 +/- 0.8 fmol/oocyte x h in water-injected oocytes to 9.2 +/- 0.65 fmol/oocyte x h (mean +/- SEM; n = 19). In contrast, [125I]T4 (4 nM) uptake was not significantly stimulated by injection of total liver mRNA. T3 uptake induced by liver mRNA was significantly inhibited by replacement of Na+ in the incubation medium by choline+ or by simultaneous incubation with 1 microM unlabeled T3. In contrast, T3 uptake by water-injected oocytes was not Na+ dependent. Fractionation of liver mRNA on a 6-20% sucrose gradient showed that maximal stimulation of T3 uptake was obtained with mRNA of 0.8-2.1 kilobases (kb). In contrast to unfractionated mRNA, the 0.7- to 2.1-kb fraction also significantly stimulated transport of T4, and it was found to induce uptake of T3 sulfate (T3S). Because T3S is a good substrate for type I deiodinase (D1), 2.3 ng rat D1 complementary RNA (cRNA) were injected either alone or together with 23 ng of the 0.8- to 2.1-kb fraction of rat liver mRNA. Compared with water-injected oocytes, injection of D1 cRNA alone did not stimulate uptake of [125I]T3S (1.25 nM). T3S uptake in liver mRNA and D1 cRNA-injected oocytes was similar to that in oocytes injected with mRNA alone, showing that transport of T3S is independent of the metabolic capacity of the oocyte. Furthermore, coinjection of liver mRNA and D1 cRNA strongly increased the production of 125I-, showing that the T3S taken up by the oocyte is indeed transported to the cell interior. In conclusion, injection of rat liver mRNA into X. laevis oocytes resulted in a stimulation of saturable, Na+-dependent T4, T3 and T3S transport, indicating that rat liver contains mRNA(s) coding for plasma membrane transporters for these iodothyronine derivatives

Uptake of triiodothyronine sulfate and suppression of thyrotropin secretion in cultured anterior pituitary cells

To investigate the uptake of triiodothyronine sulfate (T3S) and its effect on thyrotropin-releasing hormone (TRH)-induced thyrotropin (TSH) secretion, anterior pituitary cells were isolated from euthyroid rats and cultured for 3 days in medium containing 10% fetal calf serum. Incubation was performed at 37°C in medium containing 0.5% bovine serum albumin (BSA). Exposure of the pituitary cells to TRH (0.1 μmol/L) for 2 hours stimulated TSH secretion by 176%. This effect was reduced by approximately 45% after a 2-hour preincubation with T3 (0.001 to 1 μmol/L). A significant inhibitory effect of T3S on TRH-induced TSH release was only observed at a concentration of 1 μmol/L. The uptake of [125I]T3 after 1 hour of incubation was reduced by 40% ± 4% (P < .001) by simultaneous addition of 10 nmol/L unlabeled T3, whereas 1 μmol/L T3S was required to obtain a reduction of the [125I]T3 uptake by 34% ± 2% (P < .001). The amount of T3 present in the unlabeled T3S preparation was 0.25% as determined by radioimmunoassay. When pituitary cells were incubated for 1 hour with [125I]T3S or [125I]T3 both 50,000 cpm/0.25 mL), the uptake of [125I]T3zS expressed as a percentage of the dose was 0.04% ± 0.02% (mean ± SE, n = 4), whereas that of [125I]T3 amounted to 3.0% ± 0.4% (n = 4). In contrast, when hepatocytes were incubated for 1 hour with [125I]T3S, the uptake amounted to 5.1% ± 0.8% (n = 9), whereas that of [125I]T3 was 22.1% ± 1.7% (n = 9). Furthermore, [125I]T3S was as rapidly deiodinated (iodide production, 14.9% ± 2.6%; n = 9) as [125I]T3 (12.1% ± 0.8%, n = 9) by hepatocytes. It is concluded that (1) T3S is poorly taken up by pituitary cells, and (2) the suppressive effect of high concentrations of T3S on TRH-induced TSH secretion and on [125I]T3 uptake can be explained by slight contamination with T3. Thus, it appears that T3S has only a minor biological effect, if any, on the pituitary

Uptake of thyroxine in cultured anterior pituitary cells of euthyroid rats

The uptake of [125I]T4 was investigated in cultured anterior pituitary cells isolated from adult fed Wistar rats and cultured for 3 days in medium containing 10% fetal calf serum. Experiments were performed with [125I]T4 (10(5) to 2 x 10(6) cpm; 0.35-7 nM) in medium containing 0.5% or 0.1% BSA. The uptake of [125I]T4 increased with time and showed equilibrium after around 1 h of incubation. The presence of 10 microM unlabeled T4 during incubation decreased the uptake of [125I]T4 by 65-70% at all time intervals. After 24 h of incubation, 1.5% iodide and 3.2% conjugates were detected in the medium, whereas around 20% of cellular radioactivity represented [125I]T3. The 15-min uptake of [125I]T4 was significantly reduced by simultaneous incubation with 100 nM T4 (by 24%; P < 0.05), 100 nM T3 (by 38%; P < 0.001), or 10 microM rT3 (by 32%; P < 0.001), whereas 10 microM tetraiodothyroacetic acid (Tetrac) had no effect. Furthermore, preincubation (30 min) and incubation (15 min) with 10 microM monodansylcadaverine, oligomycin, or monensin reduced the uptake of [125I]T4 by 30%, 50%, and 40%, respectively (all P < 0.001). Substitution of Na+ in the buffer by K+ diminished the uptake of [125I]T4 by 39% (P < 0.005); 2 mM phenylalanine, tyrosine, or tryptophan reduced [125I]T4 uptake by 18% (P < 0.05), 18% (P = NS), and 33% (P < 0.005), respectively. Our data suggest that the pituitary contains a specific carrier-mediated energy-requiring mechanism for [125I]T4 uptake that is partly dependent on the Na+ gradient. In addition, part of [125I]T4 uptake in the pituitary might occur through an amino acid transport system. When expressed per pM of free hormone, the 15-min uptake of [125I]T4 was approximately as high as that of [125I]T3. Because the reduction of [125I]T4 uptake by T4, T3, monodansylcadaverine, oligomycin, and monensin was roughly the same as the previously reported reduction of [125I]T3 uptake by the same compounds, it is further suggested that T4 and T3 share a common carrier in cultured anterior pituitary cells

Uptake of triiodothyroacetic acid and its effect on thyrotropin secretion in cultured anterior pituitary cells

The uptake of [125I]triiodothyroacetic acid ([125I]Triac) in anterior pituitary cells was investigated and compared with that of [125I]T3. Furthermore, the effects of Triac, T3, and T4 on TSH release were compared. Cells isolated from adult male Wistar rats were cultured for 3 days in medium with 10% fetal calf serum. Uptake was measured at 37 C with [125I]Triac (100,000 cpm; 120 pM) or [125I]T3 (50,000 cpm; 50 pM) in medium with 0.5% BSA. In this medium, the ratio of the free fractions of Triac, T3, and T4 was 1:8:1. Exposure of cells to 100 nM TRH for 2 h stimulated TSH release by 80-110% (P < 0.001). Comparing total hormone levels (1 nM to 1 microM), Triac and T3 were equally effective in reducing this response, and both were 10-fold more effective than T4. The time course (15 min to 4 h) of [125I]Triac uptake was similar to that of [125I]T3, showing equilibrium after 1 h. Unlabeled Triac (1 microM) reduced the uptake of [125I]Triac and [125I]T3 at all time intervals. Expressed per pM free hormone, the cellular and nuclear uptake of [125I]Triac were twice those of [125I]T3. The 15-min uptake of [125I]Triac was reduced by incubation with 10 nM unlabeled Triac (35%; P < 0.001). Maximum inhibition (56%; P < 0.001) was found with 10 microM Triac. A similar effect was seen with 10 microM T3, T4, or 3,3',5,5'-tetraiodothyroacetic acid. Preincubation (30 min) and incubation (15 min) with 10 microM oligomycin reduced the cellular ATP content by 51% (P < 0.001), [125I]T3 uptake by 77% (P < 0.001), and [125I]Triac uptake by only 25% (P < 0.001). The temperature dependence of [125I]Triac and [125I]T3 uptake was the same. Preincubation and incubation with 10 microM monensin (reduces the Na+ gradient) or 10 microM monodansylcadaverine (inhibits receptor-mediated endocytosis) reduced 15-min [125I] Triac uptake by 15% (P < 0.005) and 19% (P < 0.005), respectively. The data show that 1) Triac, on the basis of the free hormone concentration, is more potent than T3 or T4 in suppressing TSH secretion; and 2) the rapid uptake of [125I]Triac by the anterior pituitary occurs by a carrier-mediated mechanism that is only partially dependent on ATP or the Na+ gradient

Uptake of 3,3',5,5'-tetraiodothyroacetic acid and 3,3',5'-triiodothyronine in cultured rat anterior pituitary cells and their effects on thyrotropin secretion

We compared the uptake, metabolism, and biological effects of tetraiodothyroacetic acid (Tetrac) and rT3 in anterior pituitary cells with those of T4 and T3. Cells were isolated from adult male Wistar rats and cultured for 3 days in medium with 10% fetal calf serum. Uptake was measured at 37 C in medium with 0.1% BSA for [125I]Tetrac (200,000 cpm; 240 pM) and [125I]T4 (100,000 cpm; 175 pM) or with 0.5% BSA for [125I]rT3 (100,000 cpm; 250 pM) and [125I]T3 (50,000 cpm; 50 pM). The free fraction of Tetrac was 1% that of T4 (in medium with 0.1 and with 0.5% BSA), and the free fraction of rT3 was half that of T3. Uptake of the four tracers increased sharply up to 1 h of incubation and then leveled off. Expressed as femtomoles per pM free hormone, uptake at equilibrium was 1.16 +/- 0.16 (n = 6) for Tetrac, 0.15 +/- 0.01 (n = 6) for T4, 0.023 +/- 0.003 (n = 6) for rT3, and 0.21 +/- 0.02 (n = 6) for T3. Cell-associated radioactivity after incubation for 24 h with [125I]Tetrac was represented for 15% by [125I]Triac; after incubation with [125I]T4 for 15-20% by [125I]T3, after incubation with [125I]rT3 for 6% by [125I]3,3'-T2, while [125I]T3 was still for 98% [125I]T3. Exposure of cells for 2 h to 100 nM TRH stimulated TSH release by 90-135%. Tetrac was effective in reducing this response at a free concentration of 0.05 pM, but rT3 was effective only at a free concentration of 16 nM. A free Tetrac concentration of 5 pM was equally effective as 50 pM free T4 in reducing the TSH response to TRH. In human serum, Tetrac was exclusively bound to T4-binding prealbumin. The free Tetrac fraction was 0.001% in control subjects and rose 2- to 12-fold in patients with nonthyroidal illness. As uptake of [125I]Tetrac in the pituitary was higher than that of T4 and T3, and it was more potent than T4 in reducing TSH release, Tetrac may be of potential significance for the regulation of TSH secretion in vivo