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Endocrinology Vol. 144, No. 4 1315-1323
Copyright © 2003 by The Endocrine Society

ARTICLE

Thyroid Hormone Transport by the Rat Fatty Acid Translocase

Haidy H. A. G. M. van der Putten, Edith C. H. Friesema, Nada A. Abumrad, Maria E. Everts and Theo J. Visser

Department of Veterinary Anatomy and Physiology, Utrecht University (H.H.A.G.M.v.d.P., M.E.E.), 3508 TD Utrecht, The Netherlands; Department of Internal Medicine, Erasmus University Medical Center (E.C.H.F., T.J.V.), 3000 DR Rotterdam, The Netherlands; and Department of Physiology and Biophysics, State University of New York (N.A.A.), Stony Brook, New York 11733

Address all correspondence and requests for reprints to: Theo J. Visser, Ph.D., Department of Internal Medicine, Room Ee502, Erasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: visser{at}inw3.azr.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the hypothesis that rat fatty acid translocase (rFAT) mediates the cellular uptake of T3 and other iodothyronines. Uninjected Xenopus laevis oocytes and oocytes injected 4 d previously with rFAT cRNA were incubated for 60 min at 25 C in medium containing 0.01–10 µM [125I]T3 and 0.1% BSA, or 1–100 µM [3H]oleic acid and 0.5% BSA. Injection of rFAT cRNA resulted in a 1.9-fold increase in uptake of T3 (10 nM) and a 1.4-fold increase in uptake of oleic acid (100 µM). Total T3 uptake was lower in the presence than in the absence of BSA, but relative to the free T3 concentration, uptake was increased by BSA. The fold induction of T3 uptake by rFAT was not influenced by BSA. By analyzing uptake as a function of the ligand concentration, we estimated a Km value of 3.6 µM for (total) T3 and 56 µM for (total) oleic acid. In addition to T3, rFAT mediates the uptake of T4, rT3, 3,3'-diiodothyronine, and T3 sulfate. The injection of human type III deiodinase cRNA with or without rFAT cRNA resulted in the complete deiodination of T3 taken up by the oocytes, indicating that T3 is indeed transported to the cytoplasm. In conclusion, our results demonstrate transport of T3 and other iodothyronines by rFAT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONES, most importantly T3, induce a rise in cardiac contractility and frequency (1, 2). To meet the energy requirements necessary for its work, the heart depends on the uptake of fatty acids (3) for supplying about 70% of the energy demand, whereas oxidation of carbohydrates such as glucose and lactate accounts for the remaining 30%. The contribution of fatty acids to cardiac energy production may change depending on the subject’s activity or under pathological conditions. During hyperthyroidism, fatty acid oxidation and the expression of uncoupling proteins (UCPs) in the heart are enhanced (4, 5, 6). van der Lee et al. (7) showed that UCP expression in the heart is regulated by thyroid hormones, although only in the presence of fatty acids. Fatty acid synthesis in the heart and other organs is also regulated by thyroid hormones (8). Regulation of fatty acid oxidation and synthesis is initiated by binding of T3 to nuclear T3 receptors (5, 6). Yamamoto et al. (9) showed that the binding of T3 to its receptors in nuclei isolated from heart tissue is inhibited by fatty acids.

The above findings suggest multiple interactions between thyroid hormones and fatty acids in the heart. The intracellular action and utilization of thyroid hormones and fatty acids in the heart require the transport of these compounds across the plasma membrane of the cardiomyocyte. For uptake of fatty acids, three types of transport proteins have been identified: fatty acid transport protein, plasma membrane fatty acid-binding protein, and fatty acid translocase (FAT) (10, 11, 12). FAT expression is abundant in adipose tissue, skeletal muscle, and heart (11, 13, 14), and in the latter tissue FAT was documented in vivo to facilitate a major fraction of fatty acid uptake (15, 16). FAT mRNA is up-regulated when fatty acid utilization increases (17). Furthermore, muscle contraction is acutely associated with translocation of FAT from intracellular sites to the plasma membrane (18).

FAT is an 88-kDa integral membrane protein that presumably has two membrane-spanning regions. In humans, FAT is identical to the cell surface antigen CD36 (19). In addition to fatty acid uptake, FAT/CD36 has been implicated in the binding of plasma lipoproteins and anionic phospholipids (11). For thyroid hormones, no cardiac transport protein has yet been identified, although we (20, 21, 22) and the group of Rosic et al. (23) have shown that cardiomyocytes exhibit a specific mechanism for the uptake of T3. Other groups reported the existence of such mechanisms in other cell types, including liver cells (24, 25). Interestingly, in liver cells, nonesterified fatty acids inhibit thyroid hormone uptake (26). In addition, FAT antisense mRNA expression in rat preadipocytes is associated with reduced FAT protein levels, decreased fatty acid uptake, and, moreover, inhibition of the effect of T3 on the differentiation of these cells to adipocytes (27). These observations and the high abundance of FAT in the heart prompted us to test the hypothesis that FAT mediates the uptake of T3. This appears an attractive hypothesis if it is considered that FAT transports (anionic) fatty acids and that iodothyronines are ligands for different organic anion transporters (11, 25).

Xenopus laevis oocytes have been used successfully for the functional cloning of cDNAs encoding several plasma membrane transporters (28, 29, 30, 31). We used this system to express rat FAT (rFAT) cRNA to examine the uptake of T3 and other iodothyronines and compare this with the uptake of oleic acid, a preferred fatty acid ligand for FAT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Iodothyronines were obtained from Henning (Berlin, Germany); L-phenylalanine (Phe), L-tyrosine (Tyr), estrone sulfate, 2-amino-bicyclo-(2,2,1)-heptane-carboxylic acid (BCH), oleic acid, 3-aminobenzoic acid ethyl ester (MS-222), and BSA (fraction V) were purchased from Sigma-Aldrich (St. Louis, MO). L-Tryptophan (Trp), taurocholate, and sulfobromophthalein (BSP) were obtained from Fluka (Buchs, Switzerland), and L-leucine (Leu) was purchased from Merck \|[amp ]\| Co., Inc. (Darmstadt, Germany). [125I]T3 (81.4 TBq/mmol) was purchased from NEN Life Science Products (Boston, MA), and [125I]T4 (35.6 TBq/mmol), [9,10-(N)-3H]oleic acid (296 GBq/mmol), and [3H]Trp (1.11 TBq/mmol) were purchased from Amersham International (Little Chalfont, UK). All other 125I-labeled iodothyronines were prepared as described previously (32).

RNA preparation
A 1.5-kb rFAT cDNA fragment was isolated from pSG5-rFAT (17) using EcoRI and XbaI (Promega Corp., Leiden, The Netherlands) and was subcloned into the multiple cloning region of pGEM3Z, located in between the 5'- and 3'-untranslated regions (including the polyadenylase tail) of the X. laevis ß-globin gene (33). Capped rFAT cRNA and human type III deiodinase (hD3) (34) cRNA were prepared from the cDNA clones linearized with NheI and XbaI (Promega Corp.), respectively, using the T7 RNA transcription kit (Epicentre, Madison, WI). For capping, the m7G[5']ppp[5']G cap analog was used (Epicentre). cRNAs were stored in water at -80 C.

Oocyte isolation and RNA injection
Two- to 3-yr-old X. laevis were obtained from Amrep (Breda, The Netherlands) and maintained as described previously (35, 36). Ovarian fragments were removed under MS-222 anesthesia (1 g/liter, in tap water) and hypothermia, and oocytes were prepared as described previously (35). The isolated oocytes were selected manually using morphological criteria, such as size, pigmentation, and absence of follicular debris. Healthy-looking stage V–VI oocytes (37) were transferred to six-well tissue culture plates and incubated overnight at 18 C in modified Barth’s solution [88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 0.4 mM CaCl2, 0.33 mM Ca(NO3)2, 2.4 mM NaHCO3, and 10 mM HEPES (pH 7.4)] containing 20 IU/ml penicillin and 20 µg/ml streptomycin. The next day, oocytes were injected with 23 nl water containing 4.6 ng rFAT cRNA and/or 2.3 ng hD3 cRNA using the Nanoject system (Drummond, Broomall, PA). In experiments in which uptake was examined as a function of the rFAT cRNA concentration, oocytes were injected with 23 nl water containing 1.2–9.2 ng cRNA. Uninjected oocytes were used as controls, but similar results were obtained using water-injected oocytes (data not shown). Injected and uninjected oocytes were maintained for 3–4 d at 18 C in modified Barth’s solution, with a daily change of medium.

Uptake
Uptake assays were performed as described previously (35). Four days after injection, 8–10 oocytes were incubated in 100 µl incubation medium (100 mM NaCl or 100 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM Tris, pH 7.5) supplemented with 4 x 105 cpm [125I]T3, 0.01–100 µM unlabeled T3, and 0.1% BSA or 4 x 105 cpm [3H]oleic acid, 1–100 µM unlabeled oleic acid, and 0.5% BSA. Similarly, the uptake of 10 nM 125I-labeled 3,3'-diiodothyronine (3,3'-T2), T4, and rT3 was examined in medium with 0.1% BSA, and that of 10 nM [125I]T3 sulfate ([125I]T3S) and 10 µM [3H]Trp was examined in medium without BSA. The ligand and BSA concentrations were adjusted using 5-fold concentrated stock solutions of iodothyronine and oleic acid in medium with 0.5% or 2.5% (wt/vol) BSA, respectively. To test the effect of albumin on T3 uptake, uninjected and injected oocytes were incubated with 10 nM T3 with 0%, 0.1%, or 0.5% BSA. The free T3 fraction under these conditions is 8% in the presence of 0.1% BSA and 2.5% in the presence of 0.5% BSA, as determined by equilibrium dialysis (21). The fraction of free or unbound fatty acid was calculated using a computer routine based on the association constants for oleate:BSA binding determined by Richieri et al. (38). The possible inhibitory effects of various compounds on T3 uptake were tested by adding 100 µM of these compounds to incubations containing 10 nM [125I]T3 and 0.1% BSA. After 1 h the incubation was terminated by aspiration of the incubation medium, followed by washing of the oocytes four times with 2.5 ml ice-cold Na+-containing incubation medium supplemented with 0.1% BSA. Oocytes were transferred to new tubes and analyzed individually for 125I-activity in a {gamma}-counter (Nuclear Enterprise, Edinburgh, UK). For analysis of 3H activity associated with the oocytes, individual oocytes were transferred to scintillation vials and lysed in 0.2 ml 2% (wt/vol) sodium dodecyl sulfate. Subsequently, 4 ml scintillation fluid were added (Pico-fluor, Packard, Groningen, The Netherlands), and the radioactivity was determined in a Tri-Carb 2100TR liquid scintillation analyzer (Packard).

Metabolism
Three days after injection, groups of 10 oocytes were incubated for 60 min at 25 C in medium containing 10 nM (1.5 x 106 cpm) [125I]T3 and 0.1% BSA, as described above. Before preparation for HPLC analysis, groups of oocytes were counted for radioactivity, providing uptake values comparable with those of individually analyzed oocytes. For HPLC analysis, the oocytes were homogenized in 100 µl ice-cold methanol. After centrifugation (15 min, 2500 x g, 4 C), 75 µl supernatant were mixed with 50 µl 0.02 M ammonium acetate (pH 4.0). One hundred microliters of the mixture were applied to a 4.6 x 250-mm Symmetry C18 column connected to an Alliance HPLC system (Waters Corp., Etten-Leur, The Netherlands) and eluted with a gradient of acetonitrile in 0.02 M ammonium acetate (pH 4.0) at a flow rate of 1.2 ml/min. The proportion of acetonitrile was increased in 15 min from 28% to 42%. The radioactivity in the eluate was determined using a Radiomatic A-500 flow scintillation detector (Packard, Meriden, CT).

Efflux
Groups of 10 oocytes injected with rFAT cRNA or uninjected oocytes were incubated for 60 min at 25 C in 100 µl incubation medium (see above) supplemented with 4 x 105 cpm [125I]T3, 10 nM unlabeled T3, and 0.1% BSA. One group of oocytes was processed to determine total uptake, as described above. The efflux of internalized T3 from other groups of oocytes was analyzed as follows. After removal of the incubation medium, oocytes were quickly washed with 0.5 ml Na+-containing incubation medium supplemented with 0.1% BSA, counted for 125I activity, and incubated for successive 5-min periods at 25 C with 0.5 ml incubation medium containing 0.1% BSA. After each interval, medium was rapidly replaced by fresh medium and counted for 125I activity. Radioactivity associated with the oocytes at the end of the 30-min total efflux period was counted as well. To examine the effect of extracellular T3 on T3 efflux, the efflux medium was supplemented with 10 µM unlabeled T3. The steady state efflux rate was quantified by expressing the cumulative release of 125I activity between 5 and 30 min as a percentage of that present in the oocytes at the start of the efflux period. Radioactivity released in the first 5-min period was considered to mainly represent [125I]T3 associated with the membrane of the oocyte. As extracellular unlabeled T3 did not affect the efflux of internalized [125I]T3, data from incubations with or without extracellular unlabeled T3 were combined.

Statistics and calculations
Data are presented as the mean ± SEM. Statistical significance was evaluated by repeated measures ANOVA or t test, where appropriate. Statistical significance was accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Initial experiments were carried out to confirm that rFAT, in our expression system, induced uptake of oleic acid as reported by others (19, 27, 39). Injection of rFAT cRNA increased uptake of oleic acid (100 µM in the presence of 0.5% BSA) by 1.4-fold compared with that by uninjected oocytes (P < 0.05; Fig. 1Go). Oleic acid uptake by uninjected and rFAT cRNA-injected oocytes was not inhibited by replacement of Na+ in the medium with choline (data not shown), indicating that oleic acid uptake is Na+ independent. Figure 1Go also shows uptake of 10 nM T3 in the presence of 0.1% BSA by uninjected and rFAT cRNA-injected oocytes. rFAT cRNA-injected oocytes showed a 1.9-fold induction of T3 uptake (P < 0.01), which was also Na+ independent (data not shown). Note that there was significant uptake of T3 and oleic acid in uninjected oocytes. Injection of oocytes with rFAT cRNA did not stimulate the uptake of tryptophan (data not shown).



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  		Figure 1. Oleic acid uptake and T3 uptake in uninjected oocytes ({blacksquare}) and in oocytes injected with 4.6 ng rFAT cRNA ({square}). Oocytes were incubated for 60 min at 25 C in medium supplemented with 100 µM [3H]oleic acid and 0.5% BSA or 10 nM [125I]T3 and 0.1% BSA. Data are presented as the mean ± SEM of four or nine experiments, respectively. *, P < 0.05; **, P < 0.01 (vs. uninjected oocytes).

 
Figure 2Go shows that the uptake of 10 nM T3 in medium containing 0.1% BSA, corrected for uptake in uninjected oocytes, was stimulated by the injection of increasing amounts of rFAT cRNA, reaching a near-maximum level after the injection of 4.6 ng cRNA (3.1 ± 0.2 fmol/oocyte·h). Injection of 2.3 or 4.6 ng rFAT cRNA induced the uptake of oleic acid (75 µM in the presence of 0.5% BSA) from 44.1 ± 0.9 (uninjected) to 56.3 ± 4.3 and 68.5 ± 9.2 pmol/oocyte·h, respectively (n = 2; data not shown). In all further experiments, 4.6 ng rFAT cRNA were injected per oocyte.



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  		Figure 2. Induction of 10 nM [125I]T3 uptake by oocytes after injection with 1.2–9.2 ng rFAT cRNA. Oocytes were incubated for 60 min at 25 C in medium supplemented with 0.1% BSA. Data are presented as the mean ± SEM of two or three experiments and were corrected for uptake in uninjected oocytes.

 
Figure 3Go shows the uptake of 10 nM T3 in the presence of 0.1% BSA (A) and of 75 µM oleic acid in the presence of 0.5% BSA (B) as a function of incubation time. Net uptake was calculated by subtracting uptake values measured in uninjected oocytes from those in rFAT cRNA-injected oocytes. Total and net uptake of T3 and oleic acid were linear with incubation time for at least 60 min.



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  		Figure 3. Time course of uptake of T3 (A) and oleic acid (B) by uninjected oocytes ({circ}) or oocytes injected with 4.6 ng rFAT cRNA ({bullet}). Oocytes were incubated for 5–60 min at 25 C in medium containing 10 nM [125I]T3 and 0.1% BSA or 75 µM [3H]oleic acid and 0.5% BSA. Uptake induced by the expression of rFAT cRNA was calculated by subtraction of uptake in uninjected oocytes from that observed in injected oocytes ({blacktriangleup}). The plotted lines show the result of a regression analysis of the data points using the linear function y = ax. Data are presented as the mean ± SEM of 10 oocytes in a representative experiment (of three performed).

 
From Fig. 3Go, we chose an incubation time of 60 min to measure unidirectional uptake rates of T3 (Fig. 4AGo) and oleic acid (Fig. 4BGo). T3 and oleic acid uptake through endogenous transporter(s) was determined in parallel incubations with uninjected oocytes. Uptake mediated by rFAT ({square}) was determined by subtraction of the uptake in uninjected oocytes ({blacksquare}) from that in oocytes injected with rFAT cRNA (). rFAT-induced T3 uptake was saturable, with almost complete inhibition of fractional T3 uptake at a ligand concentration of 10 µM. Endogenous T3 uptake showed only partial saturation at 10 µM T3 (Fig. 4AGo). rFAT-mediated oleic acid transport was also saturable, showing a 46% inhibition of fractional oleic acid uptake at the highest ligand concentration tested (100 µM; Fig. 4BGo). By Lineweaver-Burk analysis of the rFAT-mediated T3 uptake as a function of the total T3 concentration, we estimated an apparent Km of 3.6 µM. As in the presence of 0.1% BSA, 8% of T3 is nonprotein bound, this corresponds to an apparent Km of 0.26 µM for free T3. For (total) oleic acid, the estimated Km was 56 µM. In the presence of 0.5% (75 µM) BSA, this corresponds to a Km of 5 nM for unbound oleic acid. This value is in close agreement with that determined for oleate uptake by several cell types (11), by isolated rat heart giant sarcolemmal vesicles (40), and by CD36-transfected cells (39).



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  		Figure 4. Ligand concentration-dependent uptake of [125I]T3 (A) and [3H]oleic acid (B) by uninjected oocytes ({blacksquare}) and rFAT cRNA (4.6 ng) injected oocytes (). Oocytes were incubated for 60 min at 25 C in medium containing 0.01–10 µM [125I]T3 and 0.1% BSA, or 1–100 µM [3H]oleic acid and 0.5% BSA. Uptake induced by the expression of rFAT cRNA was calculated by subtraction of uptake in uninjected oocytes from that observed in injected oocytes ({square}). Data are expressed as a percentage of the dose per oocyte per hour and are presented as the mean ± SEM of three experiments.

 
To test the effects of albumin on T3 uptake induced by rFAT, we incubated uninjected oocytes and rFAT cRNA-injected oocytes with T3 in incubation medium with 0%, 0.1%, or 0.5% BSA. Figure 5AGo shows that increasing concentrations of BSA resulted in lower T3 uptake values in both uninjected and rFAT cRNA-injected oocytes. However, when uptake rates were expressed relative to the free T3 concentration (Fig. 5BGo), incubations with 0.1% or 0.5% BSA resulted in 4- to 5-fold higher uptake values in both uninjected and rFAT cRNA-injected oocytes. The uptake of T3 in the presence of 0%, 0.1%, or 0.5% BSA was induced 1.7-, 1.8-, and 1.8-fold, respectively, after the injection of rFAT cRNA.



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  		Figure 5. A, Effects of BSA on [125I]T3 uptake by uninjected oocytes ({blacksquare}) and oocytes injected with 4.6 ng rFAT cRNA ({square}) expressed in femtomoles per oocyte per hour (A) or femtomoles per nanomolar concentration of free T3 per oocyte per hour (B). Oocytes were incubated for 60 min at 25 C in medium containing 10 nM [125I]T3 without or with 0.1% or 0.5% BSA. Data are presented as the mean ± SEM of four experiments.

 
To examine intracellular metabolism of T3, uninjected oocytes and oocytes injected with rFAT cRNA and/or hD3 cRNA were incubated for 1 h at 25 C with 10 nM T3 and 0.1% BSA. Oocytes injected with hD3 cRNA alone showed similar T3 uptake as uninjected oocytes (3.7 ± 0.3 vs. 3.5 ± 0.2 fmol/oocyte·h). Also, oocytes injected with rFAT cRNA plus hD3 cRNA showed similar T3 uptake as oocytes injected with rFAT cRNA alone (4.5 ± 0.3 vs. 4.7 ± 0.2 fmol/oocyte·h; data not shown). The metabolism of T3 taken up by uninjected and injected oocytes was analyzed by HPLC, and the results are presented in Fig. 6Go. In oocytes injected with hD3 cRNA alone (Fig. 6CGo) or with hD3 cRNA plus rFAT cRNA (Fig. 6DGo), all T3 taken up by these oocytes was converted to 3'-monoiodothyronine (3'-T1), whereas in uninjected oocytes (Fig. 6AGo) and in oocytes injected with rFAT cRNA alone (Fig. 6BGo), T3 was not metabolized.



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  		Figure 6. HPLC chromatograms of oocyte lysate obtained from pools of 10 uninjected oocytes (A) or pools of 10 oocytes injected with 4.6 ng rFAT cRNA (B), 2.3 ng hD3 cRNA (C), or the combination of rFAT cRNA and hD3 cRNA (D) after incubation for 60 min at 25 C with 10 nM [125I]T3 and 0.1% BSA. Retention times are 5.6, 8.8, and 10.9 min for 3'-T1, 3,3'-T2, and T3, respectively.

 
In addition to thyroid hormone uptake by rFAT, we examined the possibility that FAT mediates the efflux of T3 (Fig. 7Go). Compared with uninjected oocytes there was a small, insignificant stimulation of the efflux of labeled T3. The addition of 10 µM T3 or 100 µM oleic acid to the efflux medium or increasing the BSA concentration from 0.1% to 0.5% did not affect the efflux of T3 (results not shown).



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  		Figure 7. Efflux of [125I]T3 internalized by uninjected oocytes ({circ}) or oocytes injected with 4.6 ng rFAT cRNA ({bullet}). Oocytes were incubated for 60 min at 25 C with 10 nM [125I]T3 and 0.1% BSA. After washing, the efflux of internalized [125I]T3 was determined by incubation of oocytes during successive 5-min periods at 25 C with incubation medium supplemented with 0.1% BSA. Data are presented as the mean ± SEM of four determinations in two experiments.

 
The uptakes of T3, 3,3'-T2, rT3, T4 (in the presence of 0.1% BSA), and T3S (in the absence of BSA) were determined in uninjected and rFAT cRNA-injected oocytes (Fig. 8Go). Injection of rFAT cRNA resulted in an increased uptake of all iodothyronine derivatives, which decreased in the order T3 > 3,3'-T2 > T4 {approx} T3S {approx} rT3. The uptake of Trp was not increased by the injection of rFAT cRNA (not shown), indicating that the rFAT-induced increase in iodothyronine and fatty acid uptake was not due to a nonspecific effect on the oocyte cell membrane.



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  		Figure 8. Uptake of 10 nM 125I-labeled iodothyronines by uninjected oocytes ({blacksquare}) and oocytes injected with 4.6 ng rFAT cRNA ({square}). Oocytes were incubated for 60 min at 25 C with 10 nM [125I]T3, [125I]3,3'-T2, [125I]rT, or [125I]T4 and 0.1% BSA, or 10 nM [125I]T3S without BSA. Data are the mean ± SEM of 9–10 oocytes in a representative experiment (of two performed).

 
Finally, the specificity of T3 transport mediated by rFAT was further investigated by testing the effects of 100 µM of various amino acids, BCH, taurocholate, BSP, and estrone sulfate. Except for BSP, which produced about 80% inhibition, all other compounds showed moderate effects (0–25%) on T3 transport by rFAT (Table 1Go).


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  		Table 1. Effect of various compounds on the uptake of T3

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report showing that a fatty acid transporter, rFAT, transports T3 and other iodothyronines. The uptake of T3 is Na+ independent and shows saturation at increasing ligand concentrations, with an estimated Km of 3.6 µM total T3 and 0.3 µM free T3. These values are well within the range of Km values (1.5–7 µM) reported for other transporters mediating T3 uptake after expression in oocytes (see below), although these studies were carried out in the absence of BSA (25, 41, 42, 43). In addition, we confirmed that rFAT, when expressed in X. laevis oocytes, exhibits Na+-independent oleic acid transport, as reported previously in other cell expression systems (39).

In addition to T3, rFAT transports other iodothyronines, showing a substrate preference for T3 > 3,3'-T2 > T4 {approx} rT3 as tested at a 10-nM substrate concentration in the presence of 0.1% BSA. When tested in the absence of BSA, T3S was found to be transported by rFAT. The free iodothyronine fractions in these experiments may be approximated as 100% for T3S, 8% for T3 and 3,3'-T2, 3% for rT3, and 0.8% for T4 (this study and Ref. 44). If the rFAT-mediated transport is considered relative to the approximate free hormone concentration, the substrate preference decreases in the order T4 {approx} T3 > 3,3'-T2 {approx} rT3 > T3S.

In addition to rFAT, various other transporters were shown to exhibit thyroid hormone transport activity. These include 1) the Na+-dependent organic anion transporter (NTCP), 2) members of the multispecific Na+-independent organic anion transporter (OATP) family, and 3) the L-type heterodimeric amino acid transporter, comprised of the human 4F2 heavy chain and the L amino acid transporter, LAT1 or LAT2, light chains (25). In agreement with our previous study in neonatal rat cardiomyocytes, showing that T3 uptake is Na+ independent (21), we show here that T3 transport mediated by rFAT is Na+ independent. As NTCP is a Na+-dependent transporter that is expressed exclusively in the liver, it does not play a role in T3 uptake in the heart (45). Recently, Fujiwara et al. (46) cloned a novel human organic anion transporter, OATP-E, that mediates Na+-independent uptake of T3. The mRNA encoding OATP-E is present in heart and various other tissues. The physiological relevance of thyroid hormone transport in heart by OATP-E remains to be established. In a previous study (21) we showed that T3 uptake by neonatal cardiomyocytes is not inhibited by 10–25 µM BSP, a ligand for different OATPs, including OATP-E (46). In the present study, we observed that at 100 µM BSP markedly inhibited T3 transport by rFAT. However, the other OATP ligands, estrone sulfate and taurocholate, had little or no effect on T3 uptake by rFAT, indicating a substantially different specificity of iodothyronine transport by rFAT vs. OATPs.

Ritchie et al. (47) and Friesema et al. (43) showed induction of T3 uptake by the heterodimeric system LAT expressed in X. laevis oocytes. The presence of its subunits in heart tissue (48) suggests that this transporter plays a role in T3 uptake in heart. However, BCH, the specific system L ligand, has no effect on the uptake of T3 in neonatal rat cardiomyocytes. Therefore, we concluded that the system LAT is not involved in T3 uptake in neonatal heart (21). Because T3 uptake in neonatal cardiomyocytes was partly reduced by the aromatic amino acids Trp and Tyr (20, 21), we proposed that the amino acid transport system T could be an additional uptake mechanism for T3 in these cells. Recently, a Na+-independent aromatic amino acid transporter, TAT1, has been characterized that mediates the uptake of Trp, Tyr, and Phe, but not that of T4 and T3 (49). In the present study we observed only slight effects of Trp, Tyr, Phe, and Leu on T3 transport by rFAT at a concentration (100 µM) at which these amino acids produce strong inhibition of T3 transport by LAT (43). These findings, therefore, indicate marked differences in the specificity of iodothyronine transport by rFAT vs. LAT.

Thus, a number of transporters have been identified in the heart that are capable of thyroid hormone transport in a Na+-independent manner. Results from our in vitro cell studies suggest that LAT and rOATP-E are not responsible for T3 uptake in the neonatal heart, but we cannot exclude that the results will be different in myocytes from adult rat heart. Furthermore, as yet unidentified members of these transporter families may be present in the heart and exhibit T3 transport. The 1.9-fold increase in T3 uptake after the injection of oocytes with rFAT cRNA is somewhat higher than the increases observed with other transporters characterized under the same conditions in our laboratory, i.e. 1.7-fold for rNTCP (50), 1.7-fold for rOATP1 (50), and 1.5-fold for human LAT (43). Together with the abundant expression of rFAT in rat heart (13, 14), these results suggest that rFAT is an important transporter for T3 uptake in the heart. However, the physiological relevance in vivo of rFAT and other transporters for cardiac T3 uptake remains to be established. For rFAT, this question may be addressed in animal models, in which FAT expression is impaired, e.g. CD36/FAT null mice (15), and spontaneously hypertensive rats (51). These models showed defective myocardial fatty acid uptake, whereas fatty acid uptake was increased in a transgenic mouse model with muscle-targeted overexpression of FAT (52). In addition, CD36 deficiency in humans may underlie defective myocardial fatty acids uptake and some cases of heart disease (16). In addition to these models, the primary culture of neonatal rat cardiomyocytes has proven to be a useful model in the examination of thyroid hormone transport (20, 21, 22). We will continue to examine the physiological relevance of thyroid hormone uptake by fatty acid transport mechanisms in this model and in the embryonic heart cell line H9c2 (53, 54).

We observed marked oleic acid uptake in uninjected oocytes, which is explained by the presence of endogenous FATs (55). Similar results were found for thyroid hormone uptake by native oocytes in the present and earlier studies (35, 36). Attempts to find specific inhibitors of these endogenous transporters to diminish the background were not successful. The use of the water-soluble synthetic ligand, T3 sulfamate, was attractive, because it combines a low uptake in uninjected oocytes with a similarly high induction as T3 itself after injection of rat liver mRNA (56). Unfortunately, neonatal cardiomyocytes showed no uptake of this ligand (our unpublished results). Interestingly, rFAT failed to transport T3 sulfamate (not shown). Therefore, we decided to continue our studies with T3.

The relatively high Km values for iodothyronine transport by rFAT, rOATPs, and rNTCP (see above) indicates that these transporters will not be saturated by even supraphysiological serum thyroid hormone concentrations. However, it does not suggest that these transporters have a negligible contribution to tissue thyroid hormone uptake, as this is also determined by the level of their expression, which is particularly abundant for FAT in the heart, skeletal muscle, and adipose tissue (11, 13, 14). It could also be argued that the increase in iodothyronine uptake after injection of oocytes with rFAT cRNA may be due to stimulation of the expression of an endogenous transporter rather than the expression of rFAT protein. However, we failed to detect any increase in iodothyronine transport in oocytes injected with cRNA for either one of the two subunits of the above-mentioned heterodimeric LAT, although injection of the cRNAs for both subunits produced marked stimulation of iodothyronine uptake (43). Negative responses have also been observed with cRNAs coding for a number of other candidate transporters (our unpublished observations). In addition, we found that the injection of rFAT cRNA into oocytes did not increase tryptophan transport. These findings, therefore, strongly argue against nonspecific stimulation of the expression of endogenous transporters by injection of oocytes with rFAT cRNA.

Albumin is one of the thyroid hormone-binding proteins in plasma (57). It has been shown that BSA facilitates the uptake of thyroid hormones in isolated rat hepatocytes (58, 59). Our results show no difference in the fold stimulation of T3 uptake induced by rFAT in the presence or absence of 0.1% or 0.5% BSA. Furthermore, the results showed that total T3 uptake decreased with increasing BSA, in agreement with the general idea that the unbound hormone determines the rate of uptake (60, 61). However, when we analyzed the data relative to the free hormone concentration, which decreases with increasing concentration of BSA, T3 uptake was higher in incubations with BSA than in incubations without BSA. These findings may be explained as follows. With BSA present in the water layer around the oocyte, the free T3 concentration is buffered, providing a constant pool of T3 available for uptake. Furthermore, the addition of BSA prevents the loss of thyroid hormones and fatty acids by adsorption to assay tubes and pipette walls (61).

T3 uptake mediated by rFAT is linear during the first 60 min, suggesting that uptake represents more than just binding of T3 to the plasma membrane. Metabolism of T3 by an intracellular enzyme would represent unequivocal proof that T3 enters the oocytes by rFAT expression on the cell membrane. T3 is not metabolized by native oocytes, and, thus, hD3 cRNA was injected to induce the expression of a T3-metabolizing enzyme in the oocyte. hD3 converts T3 initially to 3,3'-T2 and subsequently to 3'-T1 (62). The results show that in oocytes injected with rFAT plus hD3 cRNA or with hD3 cRNA alone, T3 was completely converted to 3'-T1, whereas in oocytes injected with rFAT cRNA alone no conversion was observed. Together with the finding that an increase in T3 uptake was only observed in oocytes injected with rFAT cRNA, this indicates that T3 transport mediated by rFAT is independent of the metabolic capacity of the oocyte and is rate-limiting for entry and metabolism of T3.

In addition to T3 uptake by rFAT, we tested the possibility that rFAT mediates the efflux of T3. Our results show only a minor increase in the efflux of labeled T3, which is not significantly different from that observed in uninjected oocytes. Furthermore, efflux was not stimulated by exchange with extracellular unlabeled T3 or oleic acid. Therefore, we conclude that rFAT does not importantly contribute to the efflux of T3 and thus is mainly involved in thyroid hormone uptake.

In summary, this report shows that rFAT exhibits Na+-independent T3 uptake with an estimated Km of 3.6 µM total T3 and 0.28 µM free T3; this was further evidenced by the complete conversion of T3 into 3'-T1. Furthermore, rFAT transports other iodothyronines, such as 3,3'-T2, T4, T3S, and rT3. We also confirmed the function of rFAT as a fatty acid transporter by inducing uptake of oleic acid. We are currently examining the physiological implications of T3 uptake in heart via fatty acid transport mechanisms in neonatal rat cardiomyocytes and H9c2 cells.


   Acknowledgments
 
The authors thank Dr. Takaaki Abe (Department of Neurophysiology, Tohoku University School of Medicine, Sendai, Japan) for his generous gift of pGEM3Z plasmid, and Prof. Dr. P. Reed Larsen (Peter Bent Brigham and Womans Hospital, Harvard Medical School, Boston, MA) for his generous gift of hD3 cDNA.


   Footnotes
 
This work was supported by The Netherlands Heart Foundation (Grant 96.175) and The Netherlands Organization for Scientific Research (Grant 903.40.194).

Abbreviations: BCH, 2-Amino-bicyclo-(2,2,1)-heptane-carboxylic acid; BSP, sulfobromophthalein; hD3, human type III deiodinase; LAT, L amino acid transporter; NTCP, Na+-dependent organic anion transporter; OATP, organic anion transporter; rFAT, rat fatty acid translocase; 3'-T1, 3'-monoiodothyronine; 3,3'-T2, 3,3'-diiodothyronine; T3S, T3 sulfate; UCP, uncoupling protein.

Received May 16, 2002.

Accepted for publication December 31, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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