Cellular Uptake Of T3 Research Articles

The difference in the cellular uptake of tocopherol and tocotrienol is influenced by their affinities to albumin

Vitamin E is classified into tocopherol (Toc) and tocotrienol (T3) based on its side chains. T3 generally has higher cellular uptake than Toc, though the responsible mechanism remains unclear. To elucidate this mechanism, we hypothesized and investigated whether serum albumin is a factor that induces such a difference in the cellular uptake of Toc and T3. Adding bovine serum albumin (BSA) to serum-depleted media increased the cellular uptake of T3 and decreased that of Toc, with varying degrees among α-, β-, γ-, and δ-analogs. Such enhanced uptake of α-T3 was not observed when cells were incubated under low temperature (the uptake of α-Toc was also reduced), suggesting that Toc and T3 bind to albumin to form a complex that results in differential cellular uptake of vitamin E. Fluorescence quenching study confirmed that vitamin E certainly bound to BSA, and that T3 showed a higher affinity than Toc. Molecular docking further indicated that the differential binding energy of Toc or T3 to BSA is due to the Van der Waals interactions via their side chain. Overall, these results suggested that the affinity of Toc and T3 to albumin differs due to their side chains, causing the difference in their albumin-mediated cellular uptake. Our results give a better mechanistic insight into the physiological action of vitamin E.

Functional Characterization of the Novel and Specific Thyroid Hormone Transporter SLC17A4.

Background: A recent genome-wide association study identified the SLC17A4 locus associated with circulating free thyroxine (T4) concentrations. Human SLC17A4, being widely expressed in the gastrointestinal tract, was characterized as a novel triiodothyronine (T3) and T4 transporter. However, apart from the cellular uptake of T3 and T4, transporter characteristics are currently unknown. In this study, we delineated basic transporter characteristics of this novel thyroid hormone (TH) transporter. Methods: We performed a broad range of well-established TH transport studies in COS-1 cells transiently overexpressing SLC17A4. We studied cellular TH uptake in various incubation buffers, TH efflux, and the inhibitory effects of different TH metabolites and known inhibitors of other TH transporters on SLC17A4-mediated TH transport. Finally, we determined the effect of tunicamycin, a pharmacological inhibitor of N-linked glycosylation, and targeted mutations in Asn residues on SLC17A4 function. Results: SLC17A4 induced the cellular uptake of T3 and T4 by ∼4 times, and of reverse (r)T3 by 1.5 times over control cells. The uptake of T4 by SLC17A4 was Na+ and Cl- independent, stimulated by low extracellular pH, and reduced by various iodothyronines and metabolites thereof, particularly those that contain at least three iodine moieties irrespective of the presence of modification at the alanine side chain. None of the classical TH transporter inhibitors studied attenuated SLC17A4-mediated TH transport. SLC17A4 also facilitates the efflux of T3 and T4, and to a lesser extent of 3,3'-diiodothyronine (T2). Immunoblot studies on lysates of transfected cells cultured in absence or presence of tunicamycin indicated that SLC17A4 is subject to N-linked glycosylation. Complementary mutational studies identified Asn66, Asn75, and Asn90, which are located in extracellular loop 1, as primary targets. Conclusions: Our studies show that SLC17A4 facilitates the transport of T3 and T4, and less efficiently rT3 and 3,3'-T2. Further studies should reveal the physiological role of SLC17A4 in TH regulation.

Sorafenib-Induced Changes in Thyroid Hormone Levels in Patients Treated for Hepatocellular Carcinoma.

The pathogenesis of tyrosine kinase inhibitor-induced thyroid hormone (TH) alterations are still a matter of debate. The objective of this study was to determine the effects of sorafenib on TH levels in patients with hepatocellular carcinoma (HCC) and to evaluate possible mechanisms. We performed a prospective cohort study between 2009 and 2016. This study was conducted at a tertiary referral center. This study included 57 consecutive patients with HCC who were treated with sorafenib. Thyroid-stimulating hormone (TSH) and free thyroxine (FT4) levels were measured every 6 weeks, and extensive thyroid function tests (TFTs) were measured before treatment (t0), after 6 weeks (t6), and at the end of therapy. The effect of sorafenib on TH transport by monocarboxylate transporter (MCT)8 or MCT10 was tested in transfected COS1 cells. Four patients (7%) developed thyroiditis. Among the other patients, 30% had elevation of TSH or FT4 above the normal range. Overall, between t0 and t6, mean TSH increased from 1.28 to 1.57 mU/L (P < 0.001) and mean FT4 from 18.4 to 21.2 pmol/L (P < 0.001). Simultaneously, the serum triiodothyronine (T3)/reverse triiodothyronine ratio and the (T3/thyroxine) ×100 ratio decreased. Sorafenib decreased cellular T3 uptake by MCT8 and to a lesser extent by MCT10. These in vivo data suggest that sorafenib affects TFTs on multiple levels. Our in vitro experiments suggest a possible role of sorafenib-induced inhibition of T3 transport into the cell by MCT8 and MCT10.

Synergistic Anticancer Effect of Tocotrienol Combined with Chemotherapeutic Agents or Dietary Components: A Review.

Tocotrienol (T3), unsaturated vitamin E, is gaining a lot of attention owing to its potent anticancer effect, since its efficacy is much greater than that of tocopherol (Toc). Various factors are known to be involved in such antitumor action, including cell cycle arrest, apoptosis induction, antiangiogenesis, anti-metastasis, nuclear factor-κB suppression, and telomerase inhibition. Owing to a difference in the affinity of T3 and Toc for the α-tocopherol transfer protein, the bioavailability of orally ingested T3 is lower than that of Toc. Furthermore, cellular uptake of T3 is interrupted by coadministration of α-Toc in vitro and in vivo. Based on this, several studies are in progress to screen for molecules that can synergize with T3 in order to augment its potency. Combinations of T3 with chemotherapeutic drugs (e.g., statins, celecoxib, and gefitinib) or dietary components (e.g., polyphenols, sesamin, and ferulic acid) exhibit synergistic actions on cancer cell growth and signaling pathways. In this review, we summarize the current status of synergistic effects of T3 and an array of agents on cancer cells, and discuss their molecular mechanisms of action. These combination strategies would encourage further investigation and application in cancer prevention and therapy.

Effect of Diethylstilbestrol on Thyroid Hormone Binding to Amphibian Transthyretins

Transthyretin (TTR) is responsible for a major part of the binding of thyroid hormone to proteins in the plasma in amphibian tadpoles. To characterize the binding properties of amphibian TTRs, the effects of 17 hydrophobic signaling molecules, including 6 endocrine disruptors, on 3,5,3′-l-[125I]triiodothyronine ([125I]T3) binding to plasma proteins were examined in bullfrog Rana catesbeiana tadpoles. T3 was the most potent competitive inhibitor among the 11 natural biological ligands studied, with an ID50 of 8 nM. Diethylstilbestrol (DES) was the most powerful inhibitor among the 6 endocrine disruptors studied, with an ID50 of 20 nM. Similar inhibitions of [125I]T3 binding by these compounds were obtained when purified recombinant Xenopus and Rana TTRs were analyzed. Scatchard analysis revealed that Xenopus and Rana TTRs each possessed a single class of binding site for T3, with a Kd of 262 and 1.9 nM, respectively, at 0°C. DES, at a concentration of 200 nM, induced the uptake of [125I]T3 into Rana red blood cells suspended in Rana plasma from prometamorphic stages XIII–XV, when TTR is present in plasma. DES induced the uptake of [125I]T3 into red blood cells to a lesser extent when they were suspended in Rana plasma from metamorphic climax stage XXIV, in which the level of TTR was lower than in plasma from the prometamorphic tadpoles. These results indicate that amphibian TTRs have the ability to bind DES with similar affinity to T3, the natural ligand, and raise the possibility that DES binding to TTR might induce the temporary elevation of the free concentration of plasma T3 followed by acceleration of cellular T3 uptake.

Heat shock decreases nuclear transport of 3,5,3'-triiodo-L-thyronine in clone 9 cells.

Thyroid hormone, T3, enters cells through an energy-dependent, saturable process and/or passive diffusion. Although the nucleus is the primary site of T3 action, exact mechanisms by which T3 is transported to nucleus are uncertain. In this report, initial cellular and nuclear uptake of T3 was determined using a rat liver cell line (clone 9), in which energy-dependent cellular T3 uptake was demonstrable. One to 5 mM sodium butyrate enhanced cellular T3 uptake with a concomitant increase in nuclear T3 uptake after 30 h. Increased cellular T3 uptake was associated with the increase in the maximum velocity of saturable cellular T3 uptake systems without affecting the Michaelis-Menten constant. Sodium butyrate, however, elicited a reduction in nuclear thyroid hormone receptor levels. On the other hand, heat shock (42 C for 60 min) reduced nuclear T3 uptake without affecting the Michaelis-Menten constant and maximum velocity of saturable cellular T3 uptake. Although nuclear receptor levels were reduced transiently after the heat shock, decreased nuclear T3 transport was not caused by the changes in the receptor levels. NADPH-dependent cytosolic T3 binding protein was undetectable in clone 9 cells before and after butyrate treatment or heat shock. In conclusion, sodium butyrate enhanced nuclear T3 uptake through the increase in cellular T3 uptake that is prerequisite to nuclear T3 transport. However, nuclear T3 uptake mechanisms independent of the cellular uptake system exist and are sensitive to heat shock. Nuclear receptors and cytosolic T3 binding proteins do not seem to be involved in the alteration of nuclear T3 uptake after sodium butyrate or heat shock. These findings suggest intracellular regulatory mechanisms of thyroid hormone transport.

Requisite structural characteristics for benzodiazepine inhibition of triiodothyronine uptake into a human liver cell line

Previously, we reported potent inhibition of triiodo-L-thyronine (T3) cellular uptake into a human liver cell line (HepG2) by central and peripheral receptor specific benzodiazepine (BZ) compounds and our working hypothesis of BZ's as direct competitors for the iodothyronine transporter, displacing T3 but not acting as a sunstrate for transport. In this report, we list other reported uptake inhibitors and compare them to 23 benzodiazepine receptor ligands, in their potency to inhibit cellular uptake of T3. The most potent inhibitors are restricted to the benzodiazepine class. From the BZ structure-activity relationship (SAR) for inhibition, we see that the nonfused phenyl ring may be essential for activity and the strongest relationship is seen with substitution at R2' where Cl > F > H. Substitution at R4' and hydroxyl substitution at R3 enhances potency as will alkyl groups at R1 or on the imidazole group in the 1,2-annelated series. With R7 substitution, Cl is preferred over NO2 but not necessarily H when R4' = Cl; this may reflect a slightly different orientation of the molecule with large aliphatic R1 groups and/or R4' substitution. The carbonyl at R2 in the 1,4 benzodiazepine series, enhances their potency. The resultant structure-activity relationship highlights the importance of the halogen-substituted nonfused phenyl ring and seems unique relative to other described benzodiazepine sites and/or effects.

Uptake of 3,5,3'-triiodothyronine by cultured rat hepatoma cells is inhibitable by nonbile acid cholephils, diphenylhydantoin, and nonsteroidal antiinflammatory drugs.

Cellular uptake of T3 was examined using rat H4 hepatoma cells. Uptake of [125I]T3 (10(-11) M) from serum-free medium was measured as the cell-associated counts retained by washed cells (2 X 10(6) per well). Displaceable uptake was 84% of total uptake at 2 min (2.9% of total counts). T4, tetraiodothyroacetic acid, triiodothyroacetic acid, rT3, and D-T3 were 2-5% as effective as T3 in displacing uptake. Nonequilibrium kinetics indicated a half-maximal uptake at 680 nM T3 with approximately 7 million sites per cell. Displaceable uptake was time and temperature dependent and was 73% inhibited by 2 mM KCN and 52% by 10 mM bacitracin but not by 2 mM ouabain or 10 microM cytochalasin B. Phloretin, 100 microM, inhibited uptake by 66%. T3 uptake was directly related to the free T3 concentration over the range of albumin concentrations, 0-10 g/liter. The nonbile acid cholephil compounds, bromosulfophthalein, iopanoic acid, and indocyanine green (all 100 microM) inhibited T3 uptake to 62%, 17%, and 5% of control, respectively. Taurocholate, methylaminoisobutyric acid, and oleic acid were noninhibitory. The half-inhibitory concentrations of reactive nonsteroidal antiinflammatory drugs were: meclofenamic acid (25 microM), mefenamic acid (45 microM), fenclofenac (69 microM), flufenamic acid (100 microM), and diclofenac (230 microM). Aspirin, ibuprofen, oxyphenbutazone, and phenylbutazone (all 100 microM) were noninhibitory. Diphenylhydantoin inhibited uptake to 50% at 75 microM. These findings suggest that T3 uptake by cultured rat hepatocytes is by an energy-dependent, saturable, stereo-selective mechanism that is dependent on cell membrane proteins. This mechanism appears to be shared by a number of other ligands, including nonbile acid cholephils and several nonsteroidal antiinflammatory drugs of the anthranilic and phenylacetic acid classes, as well as diphenylhydantoin. The bile acid taurocholate, oleic acid, and a probe for type A amino acid uptake were inactive. The extent to which these effects may modify expression of thyroid hormone action remains to be established.

Thyroid hormone action at the cellular level.

Thyroid hormones influence numerous physiological and biochemical functions. The expression of the hormonal effects involves several events. The interaction of T3 with nuclear receptors, and the stimulation of mRNA production appears to be a major step. Extranuclear binding of thyroid hormones could account for early responses. Plasma membrane receptors may play a role in the cellular uptake of T3 and the stimulation of amino acids and sugar transport. A direct control of oxidative phosphorylation through binding of T3 to mitochondrial binding sites has been proposed. The role of cytosolic binding proteins remains unclear. The understanding of the mode of action of thyroid hormones requires a better knowledge of the molecular events occurring at the nuclear level, and the relation between the nuclear and extranuclear binding sites in the hormonal expression.

Multiple effects of 5,5'-diphenylhydantoin on the thyroid hormone system.

The studies described above indicate the likelihood of a significant effect of DPH on cellular functions that are regulated by T3 at concentrations of DPH that occur during treatment of patients with Dilantin. Thus, it is possible that sensitive measures of cellular thyroid status in man, if available, might indicate that DPH treatment causes a mild hypothyroid state which may be partially compensated for by an activity of DPH as a partial thyroid hormone agonist. This review illustrates the complex array of interactions of DPH with multiple elements of the thyroid hormone system ranging from effects on T4 and T3 metabolism, serum protein binding, serum and cellular concentration of thyroid hormones, nuclear T3 binding and biologic actions of T3, to effects on hypothalamic and pituitary regulation of TSH. At the present time, no single biological mechanism common to these manifold interactions is apparent. Moreover, many of these interactions are not yet completely understood despite clinical and basic biological investigation for over 20 years. Continued investigation would appear fruitful especially for further understanding of hypothalamic regulation of TSH secretion, of cellular uptake of T3, and of the use of DPH as a partial thyroid hormone agonist. Finally, since DPH attenuates the effect of T3 at the level of the nuclear T3 receptor, the drug may serve as a prototype of agents that may be useful in the management of thyrotoxicosis.

Characterization of binding and uptake of 3,3',5-triido-L-thyronine in cultured mouse fibroblasts.

The binding and internalization of 3,3'-[125I] 5-triiodo-L-thyronine ([125I]T3) was studied in cultured Swiss 3T3-4 mouse fibroblasts. At 0 C, the binding of T3 to cells is saturable, reversible, and stereospecific. These results together with those of earlier fluorescence studies using rhodamine-labeled T3 demonstrate the presence of specific plasma membrane T3 receptors. At 37 C, the uptake of T3 reached a steady state after 1 h, and approximately 57 fmol T3 were specifically taken up by 10(6) cells. In other cell lines, 7, 19, and 201 fmol T3 were specifically taken up by Chinese hamster ovary cells (subclone 10001), Kirsten sarcoma virus-transformed NIH 3T3 mouse fibroblasts, and nontransformed NIH 3T3 mouse fibroblasts, respectively. Incorporation of T3 into nuclei followed similar kinetics and accounted for approximately 9% of the total cellular uptake. Equilibrium binding studies of T3 to isolated nuclei showed one class of binding sites with an apparent association constant of 5 X 10(9) M-1 and a binding capacity of 16 fmol/100 micrograms DNA. At 37 C, the internalization of T3 was nearly totally blocked by antimycin A or rotenone, inhibitors of oxidative phosphorylation. These results indicate that the uptake of T3 is an energy-dependent process. In the presence of bacitracin or monodansylcadaverine, substances that inhibit the receptor-mediated endocytosis of alpha 2-macroglobulin, the cellular uptake of T3 as well as the nuclear incorporation of T3 were inhibited in a concentration-dependent manner. The half-maximal inhibitory concentrations for the cellular uptake of T3 were 90 and 660 microM for monodansylcadaverine and bacitracin, respectively; for nuclear incorporation, they were 70 and 350 microM for monodansylcadaverine and bacitracin, respectively. These results indicate that receptor-mediated endocytotic uptake of T3 is a physiologically significant pathway.

Inhibition of the nuclear entry of 3,3',5'-triiodo-L-thyronine by monodansylcadaverine in GH3 cells.

We have recently reported that the entry of 3,3',5-triiodo-L-thyronine (T3) into mouse 3T3 fibroblasts occurs by receptor-mediated endocytosis (Cheng, S. Y., Maxfield, F. R., Robbins, J., Willingham, M. C., and Pastan, I. (1980) Proc. Natl. Acad. Sci. U. S. A. 77: 3425-3429). In this communication, we evaluated the functional significance of this mode of entry using GH3 cells, a growth hormone-producing cell line which has a high number of T3 nuclear receptors. T3-specific, saturable membrane uptake systems were demonstrated in GH3 cells. Monodansylcadaverine, an inhibitor of alpha 2-macroglobulin, epidermal growth factor, vesicular stomatitis virus, and T3 uptake in fibroblasts, blocked virtually all of the cellular uptake of T3, with a half-maximal concentration of 29 microM. Concomitant with the inhibition of the cellular uptake of T3, the accumulation of T3 into nuclei was reduced. The inhibitory effect of monodansylcadaverine on the reduction of T3 incorporation into nuclei was not due to the inhibition of binding to nuclear receptors, but probably was due to a decrease in the cytoplasmic availability of T3 as a result of inhibition of cellular entry. These results indicate that the uptake of T3 by receptor-mediated endocytosis is a physiologically significant process.

Elevated serum levels of T3 without metabolic effect in nutritionally deficient rats, attributable to reduced cellular uptake of T3.

Rats receiving a nutritionally deficient diet displayed markedly elevated serum free T3 levels but showed no increase in oxygen consumption. This was associated with greatly reduced ratios of hepatic cellular and nuclear 125I-T3 to serum 125I-T3. Kinetic data supported the conclusion that cellular uptake of T3 was decreased in the nutritionally deficient rats. The lack of metabolic effect, despite the elevated serum T3 levels, is attributable to reduced availability of serum T3 to tissue nuclear receptor sites.

Active transport of triiodothyronine (T 3) into isolated rat liver cells

Since the receptors for thyroid hormones are located within the cell [I] , these hormones have to be transported from the extracellular compartment through the plasma membrane into the cell. Only scarce and conflicting data concerning this transport mechanism are available. Both diffusion [2-4] and carrier-mediated processes [ 5-71 have been described or suggested. In view of the fact that amino acids [S] are transported through the plasma membrane by active or mediated processes, it is not likely that thyroid hormones enter the cell by diffusion only. In an attempt to gain more insight into the mechanism of cellular uptake of T3 the present study was performed. As a model we have chosen non-proliferative cultures of parenchymal cells from adult rat liver. The use of primary cultures offers a distinct advantage over freshly prepared liver cell suspensions. Cells are given time to recover from the damage associated with the isolation, such as temporary changes in the inability to concentrate amino acids [9] and the increased catabolic state during the first 24 h after isolation [lo] . In this report, we describe the basic characteristics of T3 uptake in this system. A preliminary account of this work has been publi~ed [l I].