ABSTRACT
Elevated concentrations of T3 and T4 concomitant with nonsuppressed TSH are found in both TSH-producing tumors and resistance to thyroid hormone beta (RTHß), posing a diagnostic challenge. We demonstrate here a 54-year-old female who presented with palpitations, goiter, and elevated free T4 with nonsuppressed TSH concentrations (TSH 2.2 mIU/L [normal range, NR 0.27-4.2 mIU/L] and FT4 59.08 pmol/L [NR 12.0-22.0 pmol/L]). Because magnetic resonance imaging revealed a pituitary microadenoma (4 mm), she was diagnosed with TSH-secreting pituitary adenoma and underwent transsphenoidal surgery. Pathological reports showed no tumor cells. Subsequent genetic testing revealed a pathogenic variant in the THRB gene resulting in a His435Arg amino acid substitution in the T3 receptor isoform beta 1 (TRß1), suggestive of RTHß. In vitro and ex vivo studies revealed that the His435Arg mutated TRß1 (TRß1-H435R) completely abolishes the T3-induced transcriptional activation, nuclear receptor corepressor 1 release, steroid receptor coactivator 1 recruitment, and T3-induced thyroid hormone target gene expression, confirming the pathogenicity of this variant. The identification of a pituitary microadenoma in a patient with RTHß led to a misdiagnosis of a TSH-producing tumor and unnecessary surgery. Genetic testing proved pivotal for an accurate diagnosis, suggesting earlier consideration in similar clinical scenarios.
ABSTRACT
Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood-brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3',5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain-cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain-cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof.
Subject(s)
Blood-Brain Barrier , Monocarboxylic Acid Transporters , Symporters , Triiodothyronine , Animals , Monocarboxylic Acid Transporters/metabolism , Monocarboxylic Acid Transporters/genetics , Humans , Symporters/metabolism , Symporters/genetics , Blood-Brain Barrier/metabolism , Mice , Triiodothyronine/metabolism , Triiodothyronine/analogs & derivatives , Chlorocebus aethiops , COS Cells , Zebrafish , Organic Anion Transporters/metabolism , Organic Anion Transporters/genetics , Biological Transport , Muscle Hypotonia/metabolism , Muscle Hypotonia/genetics , Mice, Knockout , Muscular Atrophy , Mental Retardation, X-LinkedABSTRACT
Early brain development depends on adequate transport of thyroid hormones (THs) from the maternal circulation to the fetus. To reach the fetal brain, THs have to cross several physiological barriers, including the placenta, blood-brain-barrier and blood-cerebrospinal fluid-barrier. Transport across these barriers is facilitated by thyroid hormone transmembrane transporters (THTMTs). Some endocrine disrupting chemicals (EDCs) can interfere with the transport of THs by THTMTs. To screen chemicals for their capacity to disrupt THTMT facilitated TH transport, in vitro screening assays are required. In this study, we developed assays for two THTMTs, organic anion transporter polypeptide 1C1 (OATP1C1) and organic anion transporter 4 (OAT4), both known to play a role in the transport of THs across barriers. We used overexpressing cell models for both OATP1C1 and OAT4, which showed an increased uptake of radiolabeled T4 compared to control cell lines. Using these models, we screened various reference and environmental chemicals for their ability to inhibit T4 uptake by OATP1C1 and OAT4. Tetrabromobisphenol A (TBBPA) was identified as an OATP1C1 inhibitor, more potent than any of the reference chemicals tested. Additionally perfluorooctanesulfonic acid (PFOS), perfluoroctanic acid (PFOA), pentachlorophenol and quercetin were identified as OATP1C1 inhibitors in a similar range of potency to the reference chemicals tested. Bromosulfophthalein, TBBPA, PFOA and PFOS were identified as potent OAT4 inhibitors. These results demonstrate that EDCs commonly found in our environment can disrupt TH transport by THTMTs, and contribute to the identification of molecular mechanisms underlying TH system disruption chemicals.
Subject(s)
Endocrine Disruptors , Organic Anion Transporters, Sodium-Independent , Organic Anion Transporters , Humans , Endocrine Disruptors/toxicity , Organic Anion Transporters/metabolism , Organic Anion Transporters/antagonists & inhibitors , Organic Anion Transporters, Sodium-Independent/metabolism , Fluorocarbons/toxicity , Thyroid Hormones/metabolism , Caprylates/toxicity , Thyroxine/metabolism , Biological Transport/drug effects , HEK293 Cells , Alkanesulfonic Acids/toxicity , AnimalsABSTRACT
Background: Monocarboxylate transporter 8 (MCT8) is the most specific thyroid hormone transporter identified to date, deficiency of which has been associated with severe intellectual and motor disability and abnormal serum thyroid function tests. However, it is presently unknown if MCT8, similar to other thyroid hormone transporters, also accepts additional substrates, and if disruption of their transport may contribute to the observed phenotype. Methods: In this study, we aimed to identify such substrates by applying liquid chromatography-mass spectrometry-based metabolome analysis in lysates of control and MCT8-overexpressing Xenopus oocytes. A subset of identified candidate substrates were validated by direct transport studies in transiently transfected COS-1 cells and human fibroblasts, which endogenously express MCT8. Moreover, transport characteristics were determined, including transport saturation and cis-inhibition potency of thyroid hormone transport. Results: Metabolome analysis identified 21 m/z ratios, corresponding to 87 candidate metabolites, with a 2.0-times differential abundance in MCT8-injected oocytes compared with controls. These metabolites included 3,5-diiodotyrosine (DIT) and several amino acids, including glutamate and glutamine. In accordance, MCT8-expressing COS-1 cells had 2.2-times lower intracellular accumulation of [125I]-DIT compared with control cells. This effect was largely blocked in the presence of 3,3',5-triiodothyronine (T3) (IC50: 2.5 ± 1.5 µM) or thyroxine (T4) (IC50: 5.8 ± 1.3 µM). Conversely, increasing concentrations of DIT enhanced the accumulation of T3 and T4. The MCT8-specific inhibitor silychristin increased the intracellular accumulation of DIT in human fibroblasts. COS-1 cells expressing MCT8 also exhibited a 50% reduction in intracellular accumulation of [125I]-3-monoiodotyrosine (MIT). In contrast, COS-1 cells expressing MCT8 did not alter the intracellular accumulation of [3H]-glutamate or [3H]-glutamine. However, studies in human fibroblasts showed a 1.5-1.9 times higher glutamate uptake in control fibroblasts compared with fibroblasts derived from patients with MCT8 deficiency, which was not affected in the presence of silychristin. Conclusions: Taken together, our results suggest that the iodotyrosines DIT and MIT can be exported by MCT8. MIT and DIT interfere with MCT8-mediated transport of thyroid hormone in vitro and vice versa. Future studies should elucidate if MCT8, being highly expressed in thyroidal follicular cells, also transports iodotyrosines in vivo.
Subject(s)
Monocarboxylic Acid Transporters , Symporters , Monocarboxylic Acid Transporters/metabolism , Humans , Animals , Symporters/metabolism , COS Cells , Chlorocebus aethiops , Fibroblasts/metabolism , Oocytes/metabolism , Xenopus laevis , Thyroid Hormones/metabolism , Biological Transport , Muscle Hypotonia/metabolism , Triiodothyronine/metabolism , MetabolomeABSTRACT
Background: Thyroid hormone signaling is essential for development, metabolism, and response to stress but declines during aging, the cause of which is unknown. DNA damage accumulating with time is a main cause of aging, driving many age-related diseases. Previous studies in normal and premature aging mice, due to defective DNA repair, indicated reduced hepatic thyroid hormone signaling accompanied by decreased type 1 deiodinase (DIO1) and increased DIO3 activities. We investigated whether aging-related changes in deiodinase activity are driven by systemic signals or represent cell- or organ-autonomous changes. Methods: We quantified liver and plasma thyroid hormone concentrations, deiodinase activities and expression of T3-responsive genes in mice with a global, liver-specific and for comparison brain-specific inactivation of Xpg, one of the endonucleases critically involved in multiple DNA repair pathways. Results: Both in global and liver-specific Xpg knockout mice, hepatic DIO1 activity was decreased. Interestingly, hepatic DIO3 activity was increased in global, but not in liver-specific Xpg mutants. Selective Xpg deficiency and premature aging in the brain did not affect liver or systemic thyroid signaling. Concomitant with DIO1 inhibition, Xpg -/- and Alb-Xpg mice displayed reduced thyroid hormone-related gene expression changes, correlating with markers of liver damage and cellular senescence. Conclusions: Our findings suggest that DIO1 activity during aging is predominantly modified in a tissue-autonomous manner driven by organ/cell-intrinsic accumulating DNA damage. The increase in hepatic DIO3 activity during aging largely depends on systemic signals, possibly reflecting the presence of circulating cells rather than activity in hepatocytes.
Subject(s)
Aging, Premature , Brain , DNA Repair-Deficiency Disorders , Liver , Animals , Mice , Aging/genetics , Aging, Premature/genetics , Brain/metabolism , DNA Repair-Deficiency Disorders/metabolism , Iodide Peroxidase/genetics , Liver/metabolism , Mice, Knockout , Thyroid Hormones/metabolismABSTRACT
Objective: Thyroid hormone (TH) transport represents a critical first step in governing intracellular TH regulation. It is still unknown whether the full repertoire of TH transporters has been identified. Members of the solute carrier (SLC) 22 family have substrates in common with the known TH transporters of the organic anion-transporting peptide family. Therefore, we screened the SLC22 family for TH transporters. Methods: Uptake of 1 nM of iodothyronines or sulfated iodothyronines in COS1 cells expressing SLC22 proteins was performed. Results: We first tested 25 mouse (m) SLC22 proteins for TH uptake and found that the majority of the organic anion transporter (OAT) clade were capable of 3,3',5-triiodothyronine and/or thyroxine (T4) transport. Based on phylogenetic tree analysis of the mouse and human (h) SLC22 family, we selected eight hSLC22s that grouped with the newly identified mouse TH transporters. Of these, four tested positive for uptake of one or more substrates, particularly hSLC22A11 showed robust (3-fold over control) uptake of T4. Uptake of sulfated iodothyronines was strongly (up to 17-fold) induced by some SLC22s, most notably SLC22A8, hSLC22A9, mSLC22A27 and mSLC22A29. Finally, the zebrafish orthologues of SLC22A6/8 drOatx and drSlc22a6l also transported almost all (sulfated) iodothyronines tested. The OAT inhibitors lesinurad and probenecid inhibited most SLC22 proteins. Conclusions: Our results demonstrated that members of the OAT clade of the SLC22 family constitute a novel, evolutionary conserved group of transporters for (sulfated) iodothyronines. Future studies should reveal the relevance of these transporters in TH homeostasis and physiology.
Subject(s)
Organic Anion Transporters , Zebrafish , Humans , Mice , Animals , Phylogeny , Zebrafish/metabolism , Sulfates/metabolism , Thyroid Hormones , Membrane Transport Proteins/genetics , Organic Anion Transporters/geneticsABSTRACT
Background: Fetal development is crucially dependent on thyroid hormone (TH). Maternal-to-fetal transfer of TH is a prerequisite for fetal TH availability, particularly in the first half of pregnancy. The mechanisms of transplacental transport of TH, however, are yet poorly understood. We, therefore, investigated the TH transport processes across human placentas using an ex vivo perfusion system. Methods: Intact cotyledons from term placentas of uncomplicated pregnancies were cannulated within 30 minutes after delivery and the maternal and fetal circulations were re-established. One hundred nanomolar thyroxine (T4) was added to either the maternal or fetal circulation and perfusions run up to three hours during which samples were taken from both circulations at different time points. Variables included addition of iopanoic acid (IOP) to block activity of the deiodinase type 3 (D3) and bovine serum albumin (BSA) to trap released T4. T4 and 3,3',5'-triiodothyronine concentrations in the perfusates were measured by radioimmunoassays. Results: Maternal-to-fetal transfer was slow, with T4 barely detectable in the fetal circulation unless D3 was blocked by IOP. Fetal T4 was detected after three hours perfusion (10.6 ± 0.6 nM) when BSA (34 g/L) was added in the fetal circulation to trap the released T4. In contrast, fetal-to-maternal transfer of T4 was rapid and maternal T4 increased to 43.6 ± 5.5 nM. Conclusions: Maternal-to-fetal T4 transport is limited, whereas fetal-to-maternal transport is rapid indicating that T4 transport across human term placenta is an asymmetrical process. With the high D3 activity, our observations are compatible with a protective role of the placental barrier. Future studies should reveal how the placenta exerts its gatekeeper function in ensuring optimal TH passage to the fetus.
Subject(s)
Placenta , Thyroxine , Pregnancy , Humans , Female , Triiodothyronine , Thyroid Hormones , FetusABSTRACT
Thyroid hormone is essential for fetal (brain) development. Plasma membrane transporters control the intracellular bioavailability of thyroid hormone. In the past few decades, 15 human thyroid hormone transporters have been identified, and among them, mutations in monocarboxylate transporter (MCT)8 and organic anion transporting peptide (OATP)1C1 are associated with clinical phenotypes. Different animal and human models have been employed to unravel the (patho)-physiological role of thyroid hormone transporters. However, most studies on thyroid hormone transporters focus on postnatal development. This review summarizes the research on the thyroid hormone transporters in pregnancy and fetal development, including their substrate preference, expression and tissue distribution, and physiological and pathophysiological role in thyroid homeostasis and clinical disorders. As the fetus depends on the maternal thyroid hormone supply, especially during the first half of pregnancy, the review also elaborates on thyroid hormone transport across the human placental barrier. Future studies may reveal how the different transporters contribute to thyroid hormone homeostasis in fetal tissues to properly facilitate development. Employing state-of-the-art human models will enable a better understanding of their roles in thyroid hormone homeostasis.
Subject(s)
Organic Anion Transporters , Symporters , Animals , Female , Humans , Pregnancy , Monocarboxylic Acid Transporters/genetics , Monocarboxylic Acid Transporters/metabolism , Symporters/genetics , Placenta/metabolism , Thyroid Hormones/metabolism , Fetal Development , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolismABSTRACT
Background: Fetal brain development in the first half of pregnancy is dependent on maternal thyroid hormone (TH), highlighting the importance of trans-placental TH transport. It is yet unclear which transporters are involved in this process. We aimed to identify the major TH transporters in a human placental cell model (BeWo cells). Methods: Messenger RNA expression of the known TH transporters (the monocarboxylate transporter [MCT]8, MCT10, the L-type amino acid transporter [LAT]1, LAT2, the organic anion transporting peptide [OATP]1A2 and OATP4A1) in BeWo cells and human placenta were determined by quantitative PCR. To determine the specificity and efficacy of transporter inhibitors, we first determined TH uptake at different inhibitor concentrations in African green monkey kidney fibroblast-like cells (COS1 cells) overexpressing TH transporters. We then tested TH uptake in BeWo cells in the presence or absence of the optimal inhibitor concentrations. Results: All tested TH transporters were expressed in human term placentas, whereas MCT8 was absent in BeWo cells. Both 2-amino-2-norbornanecarboxylic acid (BCH) and L-tryptophan at 1 mM inhibited LATs, whereas at the highest concentration (10 mM) L-tryptophan also inhibited MCT10. Verapamil inhibited OATP1A2 and less efficiently both MCTs, but not LATs. Both rifampicin and naringin reduced OATP1A2 activity. Finally, silychristin inhibited MCT8 at submicromolar concentrations and OATP1A2 partially only at the highest concentration tested (10 µM). In BeWo cells, verapamil reduced triiodothyronine (T3) uptake by 24%, BCH by 31%, and 1 mM L-tryptophan by 41%. The combination of BCH and verapamil additively decreased T3 uptake by 53% and the combination of BCH and 10 mM L-tryptophan by 60%, suggesting a major role for MCT10 and LATs in placental T3 uptake. Indeed, transfection of BeWo cells with MCT10-specific small interfering RNA significantly reduced T3 uptake. Only the combination of BCH and verapamil significantly reduced thyroxine (T4) uptake in BeWo cells, by 32%. Conclusions: Using pharmacological inhibitors, we show that MCT10 and LATs play a major role in T3 uptake in BeWo cells. T4 uptake appears independent of known TH transporters, suggesting the presence of, currently unknown, alternative transporter(s).
Subject(s)
Amino Acid Transport Systems, Neutral , Symporters , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Animals , Chlorocebus aethiops , Female , Humans , Monocarboxylic Acid Transporters/genetics , Monocarboxylic Acid Transporters/metabolism , Peptides/metabolism , Placenta/metabolism , Pregnancy , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Rifampin/metabolism , Symporters/metabolism , Thyroid Hormones/metabolism , Thyroxine/metabolism , Thyroxine/pharmacology , Triiodothyronine/metabolism , Triiodothyronine/pharmacology , Tryptophan/metabolism , Verapamil/metabolismABSTRACT
Background: In contrast to the thyroid hormones (THs) 3,3',5-triiodothyronine (T3) and 3,3',5,5'-tetraiodothyronine (thyroxine or T4), the binding characteristics of the thyroid hormone distributor proteins (THDP), thyroxine-binding globulin (TBG), albumin, and transthyretin in relation to TH metabolites are mostly lacking. In this study, we determined the distribution and binding affinity of TH metabolites to THDP, which is important for adequate interpretation of TH metabolite concentrations. Methods: Distribution of 125I-3,3'-diiodothyronine (3,3'-T2), -T3, -3,3',5'-triiodothyronine (rT3), -3,3',5-triiodothyroacetic acid (TA3), and -3,3',5,5'-tetraiodothyroacetic acid (TA4) to TBG, transthyretin, and albumin was determined by agar gel electrophoresis. The rank order of affinity (IC50) of TBG and transthyretin to thyronine (T0), 3-monoiodothyronine (3-T1), 3,5-diiodothyronine (3,5-T2), 3,3'-T2, T3, rT3, T4, TA3, and TA4 was determined with a radioligand, competitive binding assay. In healthy subjects, associations of serum TBG, transthyretin, and albumin with TH and its metabolites were analyzed using multiple linear regression models, adjusted for sex and age. Results: While T3 and T4 are predominantly bound to TBG, we demonstrated that the predominant THDP of 3,3'-T2 and rT3 is albumin, of TA3 is transthyretin and albumin, and of TA4 is transthyretin. With the radioligand binding assay, we showed that the rank order of affinity was T4>TA4 = rT3>T3>TA3 = 3,3'-T2 > 3-T1 = 3,5-T2>T0 for TBG (IC50-range: 0.36 nM to >100 µM) and TA4>T4 = TA3>rT3>T3 > 3,3'-T2 > 3-T1 > 3,5-T2>T0 for transthyretin (IC50-range: 0.94 nM to >100 µM). TBG, transthyretin, and albumin were not associated with T0, 3-T1, 3,3'-T2, rT3, and TA4. Conclusions: Differences in serum TBG, transthyretin, and albumin concentrations within the reference interval do not influence serum concentrations of T0, 3-T1, 3,3'-T2, rT3, and TA4. Distribution of TH metabolites between THDP differs from T4 and T3, which predominantly bind to TBG. The results from our study have potential clinical importance for adequate interpretation of TH metabolism in (patho)physiology.
Subject(s)
Prealbumin , Triiodothyronine , Humans , Reference Values , Thyroid Hormones , ThyroxineABSTRACT
OBJECTIVE: While most of the variation in thyroid function is determined by genetic factors, single nucleotide polymorphisms (SNPs) identified via genome-wide association analyses have only explained ~5% to 9% of this variance so far. Most SNPs were in or nearby genes with no known role in thyroid hormone (TH) regulation. Therefore, we performed a large-scale candidate gene study investigating the effect of common genetic variation in established TH regulating genes on serum thyrotropin [thyroid-stimulating hormone (TSH)] and thyroxine (FT4) concentrations. METHODS: SNPs in or within 10 kb of 96 TH regulating genes were included (30 031 TSH SNPs, and 29 962 FT4 SNPs). Associations were studied in 54 288 individuals from the ThyroidOmics Consortium. Linkage disequilibrium-based clumping was used to identify independently associated SNPs. SNP-based explained variances were calculated using SumHer software. RESULTS: We identified 23 novel TSH-associated SNPs in predominantly hypothalamic-pituitary-thyroid axis genes and 25 novel FT4-associated SNPs in mainly peripheral metabolism and transport genes. Genome-wide SNP variation explained ~21% (SD 1.7) of the total variation in both TSH and FT4 concentrations, whereas SNPs in the 96 TH regulating genes explained 1.9% to 2.6% (SD 0.4). CONCLUSION: Here we report the largest candidate gene analysis on thyroid function, resulting in a substantial increase in the number of genetic variants determining TSH and FT4 concentrations. Interestingly, these candidate gene SNPs explain only a minor part of the variation in TSH and FT4 concentrations, which substantiates the need for large genetic studies including common and rare variants to unravel novel, yet unknown, pathways in TH regulation.
Subject(s)
Thyroid Gland , Thyrotropin , Genome-Wide Association Study , Humans , Polymorphism, Single Nucleotide , Thyroid Gland/physiology , Thyroid Hormones , ThyroxineABSTRACT
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 â¼4 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.
Subject(s)
Genome-Wide Association Study , Thyroxine , Humans , Membrane Transport Proteins , Sodium-Phosphate Cotransporter Proteins, Type I , Thyroid Hormones/metabolism , Thyroxine/metabolism , Triiodothyronine/metabolism , TunicamycinABSTRACT
Background: In contrast to the thyroid hormones (TH) 3,3',5-triiodothyronine (T3) and thyroxine (T4), current literature on thyroid hormone metabolite concentrations in the hypothyroid and hyperthyroid states is inconclusive. It is unknown how thyroidectomy affects thyroid hormone metabolite concentrations and if levothyroxine (LT4) replacement therapy after thyroidectomy restores thyroid hormone metabolite concentrations in those without a thyroid gland. The treatment of patients with differentiated thyroid cancer (DTC) covers the euthyroid, hypothyroid, and (subclinical) hyperthyroid states and therefore provides a unique model to answer this. Here, we prospectively studied nine TH and its metabolites (THM) across different thyroid states in a cohort of patients treated for DTC. Also, three potentially important determinants for THM concentrations were studied. Methods: We prospectively included patients aged 18 to 80 years who were scheduled for DTC treatment at the Erasmus MC. Peripheral blood samples were obtained before surgery (euthyroid, endogenous TH production), after surgery just before radioactive iodine therapy (hypothyroid), and six months later on LT4 therapy ([subclinically] hyperthyroid, exogenous T4 supplementation). Nine THMs were quantified in serum with an established liquid chromatography/tandem mass spectrometry method. Repeated measurement analysis was used to compare the three different thyroid states with each other for each THM, while linear regression was used to determine the association between THM concentrations and age, sex, and kidney function. Results: In total, 77 patients (mean age 49 years; 65% women) were eligible for the study. 3,5-diiodothyronine and 3,3',5-triiodothyroacetic acids were below the lower limit of detection. Compared with the euthyroid state, all THMs were significantly decreased in the hypothyroid state and significantly increased in the (subclinically) hyperthyroid state, with T3 concentrations remaining within the reference interval. Higher age was associated with higher 3-monoiodothyronine (3-T1) concentrations (p < 0.001). Women had higher L-thyronine concentrations than men (p = 0.003). A better kidney function was associated with lower 3-T1 concentrations (p < 0.001). Conclusions: All THMs decrease after a thyroidectomy and increase under thyrotropin (TSH)-suppressive LT4-therapy, suggesting that formation of thyroid hormone metabolites is dependent on peripheral extrathyroidal metabolism of T4. This is also reflected by T3 concentrations that remained within the reference interval in patients receiving TSH-suppressive LT4-therapy as T3 has some thyroidal origin.
Subject(s)
Thyroid Gland/metabolism , Thyroxine/blood , Triiodothyronine/analogs & derivatives , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Hyperthyroidism/metabolism , Hypothyroidism/metabolism , Male , Middle Aged , Prospective Studies , Thyroid Neoplasms/metabolism , Triiodothyronine/blood , Young AdultABSTRACT
North-Eastern Brazil saw intensive application of the insecticide pyriproxyfen (PPF) during the microcephaly outbreak caused by the Zika virus (ZIKV). ZIKV requires the neural RNA-binding protein Musashi-1 to replicate. Thyroid hormone (TH) represses MSI1. PPF is a suspected TH disruptor. We hypothesized that co-exposure to the main metabolite of PPF, 4'-OH-PPF, could exacerbate ZIKV effects through increased MSI1 expression. Exposing an in vivo reporter model, Xenopus laevis, to 4'-OH-PPF decreased TH signaling and increased msi1 mRNA and protein, confirming TH-antagonistic properties. Next, we investigated the metabolite's effects on mouse subventricular zone-derived neural stem cells (NSCs). Exposure to 4'-OH-PPF dose-dependently reduced neuroprogenitor proliferation and dysregulated genes implicated in neurogliogenesis. The highest dose induced Msi1 mRNA and protein, increasing cell apoptosis and the ratio of neurons to glial cells. Given these effects of the metabolite alone, we considered if combined infection with ZIKV worsened neurogenic events. Only at the fourth and last day of incubation did co-exposure of 4'-OH-PPF and ZIKV decrease viral replication, but viral RNA copies stayed within the same order of magnitude. Intracellular RNA content of NSCs was decreased in the combined presence of 4'-OH-PPF and ZIKV, suggesting a synergistic block of transcriptional machinery. Seven out of 12 tested key genes in TH signaling and neuroglial commitment were dysregulated by co-exposure, of which four were unaltered when exposed to 4'-OH-PPF alone. We conclude that 4'-OH-PPF is an active TH-antagonist, altering NSC processes known to underlie correct cortical development. A combination of the TH-disrupting metabolite and ZIKV could aggravate the microcephaly phenotype.
Subject(s)
Neural Stem Cells , Zika Virus Infection , Zika Virus , Animals , Mice , Pyridines , Thyroid HormonesABSTRACT
CONTEXT: Genetic variants in SLC16A2, encoding the thyroid hormone transporter MCT8, can cause intellectual and motor disability and abnormal serum thyroid function tests, known as MCT8 deficiency. The C-terminal domain of MCT8 is poorly conserved, which complicates prediction of the deleteriousness of variants in this region. We studied the functional consequences of 5 novel variants within this domain and their relation to the clinical phenotypes. METHODS: We enrolled male subjects with intellectual disability in whom genetic variants were identified in exon 6 of SLC16A2. The impact of identified variants was evaluated in transiently transfected cell lines and patient-derived fibroblasts. RESULTS: Seven individuals from 5 families harbored potentially deleterious variants affecting the C-terminal domain of MCT8. Two boys with clinical features considered atypical for MCT8 deficiency had a missense variant [c.1724A>G;p.(His575Arg) or c.1796A>G;p.(Asn599Ser)] that did not affect MCT8 function in transfected cells or patient-derived fibroblasts, challenging a causal relationship. Two brothers with classical MCT8 deficiency had a truncating c.1695delT;p.(Val566*) variant that completely inactivated MCT8 in vitro. The 3 other boys had relatively less-severe clinical features and harbored frameshift variants that elongate the MCT8 protein [c.1805delT;p.(Leu602HisfsTer680) and c.del1826-1835;p.(Pro609GlnfsTer676)] and retained ~50% residual activity. Additional truncating variants within transmembrane domain 12 were fully inactivating, whereas those within the intracellular C-terminal tail were tolerated. CONCLUSIONS: Variants affecting the intracellular C-terminal tail of MCT8 are likely benign unless they cause frameshifts that elongate the MCT8 protein. These findings provide clinical guidance in the assessment of the pathogenicity of variants within the C-terminal domain of MCT8.
Subject(s)
Intellectual Disability/pathology , Monocarboxylic Acid Transporters/genetics , Mutation , Phenotype , Symporters/genetics , Adolescent , Child , Child, Preschool , Female , Humans , Intellectual Disability/genetics , Male , PrognosisABSTRACT
Mutations in the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) result in MCT8 deficiency, characterized by severe intellectual and motor disability. The MCT8 protein is predicted to have 12 transmembrane domains (TMDs) and is expressed as monomers, homodimers, and homo-oligomers. This study aimed to delineate the mechanism of MCT8 oligomerization. Coimmunoprecipitation studies demonstrated that lithium dodecyl sulfate effectively disrupts MCT8 protein complexes, indicating the involvement of non-covalent interactions. Successive C-terminal truncations of the MCT8 protein altered the oligomerization pattern only if introduced in the N-terminal half of the protein (TMD1-6). The truncation at extracellular loop 1 (E206X) still allowed homodimerization, but completely abrogated homo-oligomerization, whereas both were preserved by the C231X mutant (at TMD2), suggesting that the minimally required oligomerization sites are located proximal of Cys231. However, mutant constructs lacking the intracellular N-terminus or TMD1 and 2 were still capable to form homo-oligomers. Therefore, other domains distal of Cys231 are also likely to be involved in the formation of extensive multidomain interactions. This hypothesis was supported by structural modeling. Despite multiple approaches, MCT8 oligomerization could not be fully abrogated unless a substantial part of the protein was removed, precluding detailed studies into its functional role. Together, our findings suggest that MCT8 oligomerization involves extensive noncovalent interactions between the N-terminal halves of MCT8 proteins. Most mutations identified in patients with MCT8 deficiency have only minor effects on MCT8 oligomerization and, thus, impaired oligomerization does not appear to be an important pathogenic mechanism.
ABSTRACT
The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.
Subject(s)
Endocrine Disruptors/toxicity , High-Throughput Screening Assays/methods , Thyroid Hormones/metabolism , Animals , Blood-Brain Barrier/metabolism , Brain/drug effects , Brain/growth & development , Drug Discovery , Endocrine Disruptors/chemistry , Humans , In Vitro Techniques , InternetABSTRACT
BACKGROUND: While thyroxine (T4), 3,3',5-triiodothyronine (T3), and 3,3',5'-triiodothyronine (rT3) have routine methods available for evaluating patients with suspected thyroid disease, appropriate methods for the measurement of other thyroid hormone metabolites (THMs) are lacking. The effects of other iodothyronines or iodothyroacetic acids are therefore less explored. To better understand the (patho)physiological role of THMs, a robust method to measure iodothyronines and iodothyroacetic acids in serum in a single analysis is needed, including associated reference intervals. METHODS: Clinical and Laboratory Standards Institute guidelines, European Medicines Agency guidelines, and the National Institute of Standards and Technology protocol were used for the method validation and reference intervals. Reference intervals were determined in 132 healthy males and 121 healthy females. Serum samples were deproteinized with acetonitrile, followed by anion-exchange solid phase extraction and analysis with LC-MS/MS, using eight 13C6-internal standards. RESULTS: The analytical method validation was performed for all nine THMs. Reference intervals (2.5th to 97.5th percentile) were determined for L-thyronine (4.9-11.3 ng/dL), 3-monoiodothyronine (0.06 --0.41 ng/dL), 3,5-diiodothyronine (<0.13 ng/dL), 3,3'-diiodothyronine (0.25--0.77 ng/dL), T3 (66.4--129.9 ng/dL), rT3 (15.0--64.1 ng/dL), T4 (4.3--10.0 µg/dL), triac/3,3',5-triiodothyroacetic acid (not detected), and tetrac/3,3',5,5'-tetraiodothyroacetic acid (2.2--27.2 ng/dL). CONCLUSIONS: A broad dynamic concentration range exists among the nine THMs. This method should help to develop a better understanding of the clinical relevance of other THMs, as well as an understanding of thyroid hormone metabolism in health and disease.
Subject(s)
Tandem Mass Spectrometry/methods , Thyroid Hormones/blood , Thyroid Hormones/metabolism , Adult , Aged , Calibration , Chromatography, Liquid , Female , Healthy Volunteers , Humans , Male , Middle Aged , Reference Values , Reproducibility of Results , Young AdultABSTRACT
Background: Thyroid hormone (TH) acts on TH receptors (TRs) and regulates gene transcription by binding of TRs to TH response elements (TREs) in target gene promoters. The transcriptional activity of TRs is modulated by interactions with TR-coregulatory proteins. Mutations in TRα cause resistance to thyroid hormone alpha (RTHα). In this study, we analyzed if, beyond reduced triiodothyronine (T3) affinity, altered interactions with cofactors or different TREs could account for the differential impaired transcriptional activity of different mutants. Methods: We evaluated four mutants derived from patients (D211G, M256T, A263S, and R384H) and three artificial mutants at equivalent positions in patients with RTHß (T223A, L287V, and P398H). The in vitro transcriptional activity was evaluated on TRE-luciferase reporters (DR4, IR0, and ER6). The affinity for T3 and interaction with coregulatory proteins (nuclear receptor corepressor 1 [NCoR1] and steroid receptor coactivator 1 [SRC1]) were also determined. Results: We found that the affinity for T3 was significantly reduced for all mutants, except for TRα1-T223A. The reduction in the T3 sensitivity of the transcriptional activity on three TREs, the dissociation of the corepressor NCoR1, and the association of the coactivator SRC1 recruitment for each mutant correlated with the reduced affinity for T3. We did not observe mutation-specific alterations in interactions with cofactors or TREs. Conclusions: In summary, the degree of impaired transcriptional activity of mutants is mainly determined by their reduced affinity for T3.
Subject(s)
Mutation/genetics , Thyroid Hormone Receptors alpha/genetics , Thyroid Hormone Receptors alpha/metabolism , Cell Line, Tumor , Computer Simulation , Humans , Ligands , Models, Molecular , Nuclear Receptor Co-Repressor 1/genetics , Nuclear Receptor Coactivator 1/genetics , Protein Binding , Response Elements , Transcription, Genetic , Triiodothyronine/metabolismABSTRACT
Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause MCT8 deficiency, characterized by severe intellectual and motor disability and abnormal serum thyroid function tests. Various Mct8 knock-out mouse models as well as mct8 knock-out and knockdown zebrafish models are used as a disease model for MCT8 deficiency. Although important for model eligibility, little is known about the functional characteristics of the MCT8 orthologues in these species. Therefore, we here compared the functional characteristics of mouse (mm) MCT8 and zebrafish (dr) Mct8 to human (hs) MCT8. Methods: We performed extensive transport studies in COS-1 and JEG-3 cells transiently transfected with hsMCT8, drMct8, and mmMCT8. Protein expression levels and subcellular localization were assessed by immunoblotting, surface biotinylation, and immunocytochemistry. Sequence alignment and structural modeling were used to interpret functional differences between the orthologues. Results: hsMCT8, drMct8, and mmMCT8 all facilitated the uptake and efflux of 3,3'-diiodothyronine (3,3'-T2), rT3, triiodothyronine (T3), and thyroxine (T4), although the initial uptake rates of drMct8 were 1.5-4.0-fold higher than for hsMCT8 and mmMCT8. drMct8 exhibited 3-50-fold lower apparent IC50 values than hsMCT8 and mmMCT8 for all tested substrates, and substrate preference of drMct8 (3,3'-T2, T3 > T4 > rT3) differed from hsMCT8 and mmMCT8 (T3 > T4 > rT3, 3,3'-T2). Compared with hsMCT8 and mmMCT8, cis-inhibition studies showed that T3 uptake by drMct8 was inhibited at a lower concentration and by a broader spectrum of TH metabolites. Total and cell surface expression levels of drMct8 and hsMCT8 were equal and both significantly exceeded those of mmMCT8. Structural modeling located most non-conserved residues outside the substrate pore, except for H192 in hsMCT8, which is replaced by a glutamine in drMct8. However, a H192Q substituent of hsMCT8 did not alter its transporter characteristics. Conclusion: Our studies substantiate the eligibility of mice and zebrafish models for human MCT8 deficiency. However, differences in the intrinsic transporter properties of MCT8 orthologues may exist, which should be realized when comparing MCT8 deficiency in different in vivo models. Moreover, our findings may indicate that the protein domains outside the substrate channel may play a role in substrate selection and protein stability.