Adaptive Thermogenesis Driving Catch-Up Fat Is Associated With Increased Muscle Type 3 and Decreased Hepatic Type 1 Iodothyronine Deiodinase Activities: A Functional and Proteomic Study.

Refeeding after caloric restriction induces weight regain and a disproportionate recovering of fat mass rather than lean mass (catch-up fat) that, in humans, associates with higher risks to develop chronic dysmetabolism. Studies in a well-established rat model of semistarvation-refeeding have reported that catch-up fat associates with hyperinsulinemia, glucose redistribution from skeletal muscle to white adipose tissue and suppressed adaptive thermogenesis sustaining a high efficiency for fat deposition. The skeletal muscle of catch-up fat animals exhibits reduced insulin-stimulated glucose utilization, mitochondrial dysfunction, delayed in vivo contraction-relaxation kinetics, increased proportion of slow fibers and altered local thyroid hormone metabolism, with suggestions of a role for iodothyronine deiodinases. To obtain novel insights into the skeletal muscle response during catch-up fat in this rat model, the functional proteomes of tibialis anterior and soleus muscles, harvested after 2 weeks of caloric restriction and 1 week of refeeding, were studied. Furthermore, to assess the implication of thyroid hormone metabolism in catch-up fat, circulatory thyroid hormones as well as liver type 1 (D1) and liver and skeletal muscle type 3 (D3) iodothyronine deiodinase activities were evaluated. The proteomic profiling of both skeletal muscles indicated catch-up fat-induced alterations, reflecting metabolic and contractile adjustments in soleus muscle and changes in glucose utilization and oxidative stress in tibialis anterior muscle. In response to caloric restriction, D3 activity increased in both liver and skeletal muscle, and persisted only in skeletal muscle upon refeeding. In parallel, liver D1 activity decreased during caloric restriction, and persisted during catch-up fat at a time-point when circulating levels of T4, T3 and rT3 were all restored to those of controls. Thus, during catch-up fat, a local hypothyroidism may occur in liver and skeletal muscle despite systemic euthyroidism. The resulting reduced tissue thyroid hormone bioavailability, likely D1- and D3-dependent in liver and skeletal muscle, respectively, may be part of the adaptive thermogenesis sustaining catch-up fat. These results open new perspectives in understanding the metabolic processes associated with the high efficiency of body fat recovery after caloric restriction, revealing new implications for iodothyronine deiodinases as putative biological brakes contributing in suppressed thermogenesis driving catch-up fat during weight regain.

Unique near-complete deletion of GLI2 in a patient with combined pituitary hormone deficiency and post-axial polydactyly.

INTRODUCTION: Combined pituitary hormone deficiency (CPHD) can cause a broad spectrum of health problems, ranging from short stature only, to convulsions or even death. In the majority of patients, the cause is unknown. METHODS: The idex case had unexplained CPHD, pituitary anomalies on MRI and polydactyly. In the patients and her unaffected parents, we performed SNP array analysis and Whole Exome Sequencing, after candidate gene analysis turned out negative. RESULTS: We found a unique de novo heterozygous 229.9 kb deletion in the index case on chr. 2q14.2. This deletion covered 12 out of the 13 coding exons of the GLI2 gene, a transcription factor involved in midline formation and previously associated with CPHD. As reported GLI2 deletions and mutations show a large phenotypic variability, we performed a genotype-phenotype analysis. This revealed that GLI2 missense mutations usually present with a 'ppp-only' phenotype (pituitary anomalies ± postaxial polydactyly without brain phenotype), whereas the 'ppp-plus' phenotype (with major brain malformations and/or intellectual disabilities) is more frequent in patients with larger deletions, and those with frameshift mutations/point mutations or splice variants resulting in a stop codon (p < .001). CONCLUSION: The present case shows that a deletion of the GLI2 gene only (not affecting any of the adjacent genes) causes pituitary anomalies without brain phenotype. This suggests that brain phenotype only occurs when additional genes adjacent to GLI2 are deleted, or when mutations result in truncated GLI2 mRNA/protein. However, due to the lack of functional data for many GLI2 mutations and based on the available information regarding variable penetrance, phenotype-genotype correlations need to be made with caution.

The In Vitro Functional Impairment of Thyroid Hormone Receptor Alpha 1 Isoform Mutants Is Mainly Dictated by Reduced Ligand Sensitivity.

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.

Effectiveness and safety of the tri-iodothyronine analogue Triac in children and adults with MCT8 deficiency: an international, single-arm, open-label, phase 2 trial.

BACKGROUND: Deficiency of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) causes severe intellectual and motor disability and high serum tri-iodothyronine (T3) concentrations (Allan-Herndon-Dudley syndrome). This chronic thyrotoxicosis leads to progressive deterioration in bodyweight, tachycardia, and muscle wasting, predisposing affected individuals to substantial morbidity and mortality. Treatment that safely alleviates peripheral thyrotoxicosis and reverses cerebral hypothyroidism is not yet available. We aimed to investigate the effects of treatment with the T3 analogue Triac (3,3',5-tri-iodothyroacetic acid, or tiratricol), in patients with MCT8 deficiency. METHODS: In this investigator-initiated, multicentre, open-label, single-arm, phase 2, pragmatic trial, we investigated the effectiveness and safety of oral Triac in male paediatric and adult patients with MCT8 deficiency in eight countries in Europe and one site in South Africa. Triac was administered in a predefined escalating dose schedule-after the initial dose of once-daily 350 µg Triac, the daily dose was increased progressively in 350 µg increments, with the goal of attaining serum total T3 concentrations within the target range of 1·4-2·5 nmol/L. We assessed changes in several clinical and biochemical signs of hyperthyroidism between baseline and 12 months of treatment. The prespecified primary endpoint was the change in serum T3 concentrations from baseline to month 12. The co-primary endpoints were changes in concentrations of serum thyroid-stimulating hormone (TSH), free and total thyroxine (T4), and total reverse T3 from baseline to month 12. These analyses were done in patients who received at least one dose of Triac and had at least one post-baseline evaluation of serum throid function. This trial is registered with ClinicalTrials.gov, number NCT02060474. FINDINGS: Between Oct 15, 2014, and June 1, 2017, we screened 50 patients, all of whom were eligible. Of these patients, four (8%) patients decided not to participate because of travel commitments. 46 (92%) patients were therefore enrolled in the trial to receive Triac (median age 7·1 years [range 0·8-66·8]). 45 (98%) participants received Triac and had at least one follow-up measurement of thyroid function and thus were included in the analyses of the primary endpoints. Of these 45 patients, five did not complete the trial (two patients withdrew [travel burden, severe pre-existing comorbidity], one was lost to follow-up, one developed of Graves disease, and one died of sepsis). Patients required a mean dose of 38.3 µg/kg of bodyweight (range 6·4-84·3) to attain T3 concentrations within the target range. Serum T3 concentration decreased from 4·97 nmol/L (SD 1·55) at baseline to 1·82 nmol/L (0·69) at month 12 (mean decrease 3·15 nmol/L, 95% CI 2·68-3·62; p<0·0001), while serum TSH concentrations decreased from 2·91 mU/L (SD 1·68) to 1·02 mU/L (1·14; mean decrease 1·89 mU/L, 1·39-2·39; p<0·0001) and serum free T4 concentrations decreased from 9·5 pmol/L (SD 2·5) to 3·4 (1·6; mean decrease 6·1 pmol/L (5·4-6·8; p<0·0001). Additionally, serum total T4 concentrations decreased by 31·6 nmol/L (28·0-35·2; p<0·0001) and reverse T3 by 0·08 nmol/L (0·05-0·10; p<0·0001). Seven treatment-related adverse events (transiently increased perspiration or irritability) occurred in six (13%) patients. 26 serious adverse events that were considered unrelated to treatment occurred in 18 (39%) patients (mostly hospital admissions because of infections). One patient died from pulmonary sepsis leading to multi-organ failure, which was unrelated to Triac treatment. INTERPRETATION: Key features of peripheral thyrotoxicosis were alleviated in paediatric and adult patients with MCT8 deficiency who were treated with Triac. Triac seems a reasonable treatment strategy to ameliorate the consequences of untreated peripheral thyrotoxicosis in patients with MCT8 deficiency. FUNDING: Dutch Scientific Organization, Sherman Foundation, NeMO Foundation, Wellcome Trust, UK National Institute for Health Research Cambridge Biomedical Centre, Toulouse University Hospital, and Una Vita Rara ONLUS.

In Vitro Characterization of Human, Mouse, and Zebrafish MCT8 Orthologues.

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.

Thyroid hormone availability in the human fetal brain: novel entry pathways and role of radial glia.

Thyroid hormones (TH) are crucial for brain development; their deficiency during neurodevelopment impairs neural cell differentiation and causes irreversible neurological alterations. Understanding TH action, and in particular the mechanisms regulating TH availability in the prenatal human brain is essential to design therapeutic strategies for neurological diseases due to impaired TH signaling during neurodevelopment. We aimed at the identification of cells involved in the regulation of TH availability in the human brain at fetal stages. To this end, we studied the distribution of the TH transporters monocarboxylate transporter 8 (MCT8) and organic anion-transporting polypeptide 1C1 (OATP1C1), as well as the TH-metabolizing enzymes types 2 and 3 deiodinases (DIO2 and DIO3). Paraffin-embedded human brain sections obtained from necropsies of thirteen fetuses from 14 to 38 gestational weeks were analyzed by immunohistochemistry and in situ hybridization. We found these proteins localized along radial glial cells, in brain barriers, in Cajal-Retzius cells, in migrating fibers of the brainstem and in some neurons and glial cells with particular and complex spatiotemporal patterns. Our findings point to an important role of radial glia in controlling TH delivery and metabolism and suggest two additional novel pathways for TH availability in the prenatal human brain: the outer, and the inner cerebrospinal fluid-brain barriers. Based on our data we propose a model of TH availability for neural cells in the human prenatal brain in which several cell types have the ability to autonomously control the required TH content.

Mutated Thyroid Hormone Transporter OATP1C1 Associates with Severe Brain Hypometabolism and Juvenile Neurodegeneration.

BACKGROUND: Thyroid hormones (TH) are essential for brain development and function. The TH transporters monocarboxylate transporter 8 (MCT8) and organic anion transporter1 C1 (OATP1C1) facilitate the transport of TH across the blood-brain barrier and into glia and neuronal cells in the brain. Loss of MCT8 function causes Allan-Herndon-Dudley syndrome (AHDS, OMIM 300523) characterized by severe intellectual and motor disability due to cerebral hypothyroidism. Here, the first patient with loss of OATP1C1 function is described. The patient is a 15.5-year-old girl with normal development in the first year of life, who gradually developed dementia with spasticity and intolerance to cold. Brain imaging demonstrated gray and white matter degeneration and severe glucose hypometabolism. METHODS: Exome sequencing of the patient and parents was performed to identify the disease-causing mutation, and the effect of the mutation was studied through a panel of in vitro experiments, including thyroxine uptake studies, immunoblotting, and immunocytochemistry. Furthermore, the clinical effects of treatment with the triiodothyronine analogue triiodothyroacetic acid (Triac) are described. RESULTS: Exome sequencing identified a homozygous missense mutation in OATP1C1, changing the highly conserved aspartic acid 252 to asparagine (D252N). In vitro, the mutated OATP1C1 displays impaired plasma membrane localization and decreased cellular thyroxine uptake. After treatment with Triac, the clinical condition improved in several domains. CONCLUSIONS: This is the first report of human OATP1C1 deficiency compatible with brain-specific hypothyroidism and neurodegeneration.

Role of Leucine 341 in Thyroid Hormone Receptor Beta Revealed by a Novel Mutation Causing Thyroid Hormone Resistance.

Leucine 341 has been predicted from crystal structure as an important residue for thyroid hormone receptor beta (TRß) function, but this has never been confirmed in functional studies. Here, a novel p.L341V mutation as a cause of resistance to TRß is described, suggesting an important role for L341 in TRß function. In silico and in vitro studies confirmed that substituting L341 with valine and other non-polar amino acids impairs sensitivity of TRß for triiodothyronine to various degrees, depending on their side-chain size and orientation.

The Association of Thyroid Function With Bone Density During Childhood.

Context: Although the skeleton is a well-known thyroid hormone target organ, very little data are available on the association of thyroid function with bone outcomes during childhood. Objective: To study the association of thyroid function with bone mass during childhood. Design, Setting, and Participants: Population-based prospective cohort including 4204 children with TSH and free T4 (FT4) measured at the age of 6 years. Main Outcome Measures: Bone density was assessed by a total body dual-energy X-ray absorptiometry scan at the median age of 6 years (95% range, 5.6 to 7.9) and at the age of 10 years (95% range, 9.0 to 10.9) in 4204 and 3404 participants, respectively. Results: There was an inverse association of TSH with bone mineral density (BMD) at the age of 6 (ß -0.028 ± 0.011, P = 0.009) and with follow-up measurements at the age of 10 (ß -0.027 ± 0.011, P = 0.014), but not with bone mineral content (BMC) at the age of 6 (ß -0.028 ± 0.015, P = 0.06) or for follow-up measurements of BMC at the age of 10 (ß -0.011 ± 0.015, P = 0.47). There was an inverse association of FT4 with BMD (ß -0.016 ± 0.006, P = 0.014) and BMC (ß -0.023 ± 0.009, P = 0.009) cross-sectionally, and also at the age of 10 years (BMD: ß -0.018 ± 0.007, P = 0.007; BMC: ß -0.021 ± 0.009, P = 0.020). Conclusion: A higher FT4 concentration is associated with lower bone mass at the age of 6 and at the age of 10 years. These data provide insights into the effects of thyroid function on bone physiology during childhood.

Thyroid Hormone Transporters MCT8 and OATP1C1 Control Skeletal Muscle Regeneration.

Thyroid hormone (TH) transporters are required for the transmembrane passage of TH in target cells. In humans, inactivating mutations in the TH transporter MCT8 cause the Allan-Herndon-Dudley syndrome, characterized by severe neuromuscular symptoms and an abnormal TH serum profile, which is fully replicated in Mct8 knockout mice and Mct8/Oatp1c1 double-knockout (M/O DKO) mice. Analysis of tissue TH content and expression of TH-regulated genes indicate a thyrotoxic state in Mct8-deficient skeletal muscles. Both TH transporters are upregulated in activated satellite cells (SCs). In M/O DKO mice, we observed a strongly reduced number of differentiated SCs, suggesting an impaired stem cell function. Moreover, M/O DKO mice and mice lacking both transporters exclusively in SCs showed impaired skeletal muscle regeneration. Our data provide solid evidence for a unique gate-keeper function of MCT8 and OATP1C1 in SC activation, underscoring the importance of a finely tuned TH signaling during myogenesis.

Multiple effects of cold exposure on livers of male mice.

Cold exposure of mice is a common method to stimulate brown adipose tissue (BAT) activity and induce browning of white adipose tissue (WAT) that has beneficial effects on whole-body lipid metabolism, including reduced plasma triglyceride (TG) concentrations. The liver is a key regulatory organ in lipid metabolism as it can take up as well as oxidize fatty acids. The liver can also synthesize, store and secrete TGs in VLDL particles. The effects of cold exposure on murine hepatic lipid metabolism have not been addressed. Here, we report the effects of 24-h exposure to 4°C on parameters of hepatic lipid metabolism of male C57BL/6J mice. Cold exposure increased hepatic TG concentrations by 2-fold (P < 0.05) but reduced hepatic lipogenic gene expression. Hepatic expression of genes encoding proteins involved in cholesterol synthesis and uptake such as the LDL receptor (LDLR) was significantly increased upon cold exposure. Hepatic expression of Cyp7a1 encoding the rate-limiting enzyme in the classical bile acid (BA) synthesis pathway was increased by 4.3-fold (P < 0.05). Hepatic BA concentrations and fecal BA excretion were increased by 2.8- and 1.3-fold, respectively (P < 0.05 for both). VLDL-TG secretion was reduced by approximately 50% after 24 h of cold exposure (P < 0.05). In conclusion, cold exposure has various, likely intertwined effects on the liver that should be taken into account when studying the effects of cold exposure on whole-body metabolism.

Serum microRNA profiles in athyroid patients on and off levothyroxine therapy.

BACKGROUND: Levothyroxine replacement treatment in hypothyroidism is unable to restore physiological thyroxine and triiodothyronine concentrations in serum and tissues completely. Normal serum thyroid stimulating hormone (TSH) concentrations reflect only pituitary euthyroidism and, therefore, novel biomarkers representing tissue-specific thyroid state are needed. MicroRNAs (miRNAs), small non-coding regulatory RNAs, exhibit tissue-specific expression patterns and can be detectable in serum. Previous studies have demonstrated differential expression of (precursors of) miRNAs in tissues under the influence of thyroid hormone. OBJECTIVE: To study if serum miRNA profiles are changed in different thyroid states. DESIGN AND METHODS: We studied 13 athyroid patients (6 males) during TSH suppressive therapy and after 4 weeks of thyroid hormone withdrawal. A magnetic bead capture system was used to isolate 384 defined miRNAs from serum. Subsequently, the TaqMan Array Card 3.0 platform was used for profiling after individual target amplification. RESULTS: Mean age of the subjects was 44.0 years (range 20-61 years). Median TSH levels were 88.9 mU/l during levothyroxine withdrawal and 0.006 mU/l during LT4 treatment with a median dosage of 2.1 µg/kg. After normalization to allow inter-sample analysis, a paired analysis did not demonstrate a significant difference in expression of any of the 384 miRNAs analyzed on and off LT4 treatment. CONCLUSION: Although we previously showed an up-regulation of pri-miRNAs 133b and 206 in hypothyroid state in skeletal muscle, the present study does not supply evidence that thyroid state also affects serum miRNAs in humans.

Deafness and loss of cochlear hair cells in the absence of thyroid hormone transporters Slc16a2 (Mct8) and Slc16a10 (Mct10).

Transmembrane proteins that mediate the cellular uptake or efflux of thyroid hormone potentially provide a key level of control over neurodevelopment. In humans, defects in one such protein, solute carrier SLC16A2 (MCT8) are associated with psychomotor retardation. Other proteins that transport the active form of thyroid hormone triiodothyronine (T3) or its precursor thyroxine (T4) have been identified in vitro but the wider significance of such transporters in vivo is unclear. The development of the auditory system requires thyroid hormone and the cochlea is a primary target tissue. We have proposed that the compartmental anatomy of the cochlea would necessitate transport mechanisms to convey blood-borne hormone to target tissues. We report hearing loss in mice with mutations in Slc16a2 and a related gene Slc16a10 (Mct10, Tat1). Deficiency of both transporters results in retarded development of the sensory epithelium similar to impairment caused by hypothyroidism, compounded with a progressive degeneration of cochlear hair cells and loss of endocochlear potential. Administration of T3 largely restores the development of the sensory epithelium and limited auditory function, indicating the T3-sensitivity of defects in the sensory epithelium. The results indicate a necessity for thyroid hormone transporters in cochlear development and function.

Effects of Chemical Chaperones on Thyroid Hormone Transport by MCT8 Mutants in Patient-Derived Fibroblasts.

Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) result in severe intellectual and motor disability. At present, no effective therapy is available to restore TH signaling in MCT8-dependent tissues. Recent in vitro studies in stable overexpression cell models suggested that the function of certain mutant MCT8 proteins, specifically those that affect protein stability and intracellular trafficking (e.g., p.F501del), could be partially recovered by chemical chaperones. However, the effects of chaperones have not been demonstrated in other commonly used models for MCT8 deficiency, including transient overexpression models and patient-derived fibroblasts. Here, we demonstrate that the chemical chaperone 4-phenylbutyric acid (PBA) similarly potentiates the T3 transport function of wild-type and p.F501del mutant MCT8 in transiently transfected COS-1 cells by increasing MCT8 messenger RNA, total protein, and cell surface expression levels. Although PBA also increased the cell surface expression levels of the p.R445L mutant, no functional improvement was observed, which is in line with the proposed important role of Arg445 in substrate translocation. In contrast, PBA showed only minimal effects in ex vivo studies using control or p.F501del patient-derived fibroblasts. Moreover, the MCT8-specific inhibitor silychristin did not change these minimal effects, suggesting that the underlying mechanism is unrelated to the rescue of functional MCT8. Together, these findings indicate that the potency of chaperones to rescue mutant MCT8 function strongly depends on the cellular model and stress the need for further preclinical studies before clinically available chaperones should be considered as a treatment option in patients with MCT8 deficiency.

Effects of Thyrotropin on Peripheral Thyroid Hormone Metabolism and Serum Lipids.

BACKGROUND: Subclinical hypothyroidism is associated with dyslipidemia and atherosclerosis. Whether these effects are in part mediated via direct effects of thyrotropin (TSH) on peripheral thyroid hormone (TH) metabolism and/or concentrations of serum lipids is not clear. OBJECTIVE: This study examined whether TSH has direct effects on peripheral TH metabolism and serum lipids. METHODS: Eighty-two patients with differentiated thyroid cancer were retrospectively analyzed. All patients had undergone total thyroidectomy and 131I remnant ablation. During follow-up, two successive injections of recombinant human TSH (rhTSH) were administered to patients on a stable dose of levothyroxine. In all patients, TSH, thyroxine (T4), free T4 (fT4), triiodothyronine (T3), reverse T3 (rT3), total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, apolipoprotein B, lipoprotein(a), and triglyceride levels were measured immediately before the first and approximately 72 hours after the second injection of rhTSH. RESULTS: After rhTSH stimulation, T3 values decreased (from 1.91 to 1.81 nmol/L; p < 0.001). T4, fT4, and rT3 did not change. After rhTSH, median apolipoprotein B increased from 0.90 to 0.92 g/L (p = 0.03), lipoprotein(a) from 0.21 to 0.24 g/L (p < 0.001), and triglycerides from 1.98 to 2.50 mmol/L (p < 0.001). Serum high-density lipoprotein cholesterol decreased from 0.98 to 0.81 mmol/L (p < 0.001). Multiple regression analysis showed that the changes in lipids were most closely associated with the decrease in T3 levels. CONCLUSIONS: TSH has direct effects on peripheral TH metabolism by decreasing T3 levels in levothyroxine-treated thyroidectomized patients. This decrease in T3 levels is accompanied by unfavorable changes in serum lipids.

Dose Dependency and a Functional Cutoff for TPO-Antibody Positivity During Pregnancy.

Objective: To investigate a dose dependency of thyroperoxidase antibody (TPOAb) concentrations in relation to thyroid function and premature delivery and define a population-based, pregnancy-specific, functional cutoff for TPOAb positivity. Design: Individual participant meta-analysis of three prospective birth cohorts: the Amsterdam Born Children and their Development study, and the Holistic Approach to Pregnancy. Setting: Population-based studies in the Netherlands (2002 to 2014). Participants: A total of 11,212 pregnant women (<20 weeks' gestation). Main Outcome Measures: Thyrotropin (TSH) and FT4 concentrations, premature delivery. Results: In all cohorts, there was a dose-dependent positive association of TPOAb concentrations with TSH concentrations, as well as a dose-dependent negative association with FT4 concentrations during early pregnancy (all P < 0.0001). There was a dose-dependent association of TPOAb concentrations with the risk of premature delivery, which was also modified by TSH concentrations. Women with TPOAb concentrations from the 92nd percentile upward had a higher TSH and a higher risk of a TSH >2.5 mU/L (range, 19.4% to 51.3%). Stratified analyses showed that women with TPOAb concentrations below manufacturer cutoffs already had a higher risk of premature delivery, especially when TSH concentrations were high or in the high-normal range. Conclusions: This study demonstrated a dose-dependent relationship between TPOAbs and thyroid function as well as the risk of premature delivery. Furthermore, our results indicate that the currently used cutoffs for TPOAb positivity may be too high. Furthermore, the use of a population-based cutoff for TPOAbs may identify women with a clinically relevant extent of thyroid autoimmunity and a higher risk of premature delivery but that would not be considered TPOAb positive or eligible for treatment otherwise.

Thyroid State Regulates Gene Expression in Human Whole Blood.

Context: Despite the well-recognized clinical features resulting from insufficient or excessive thyroid hormone (TH) levels in humans, it is largely unknown which genes are regulated by TH in human tissues. Objective: To study the effect of TH on human gene expression profiles in whole blood, mainly consisting of T3 receptor (TR) α-expressing cells. Methods: We performed next-generation RNA sequencing on whole blood samples from eight athyroid patients (four females) on and after 4 weeks off levothyroxine replacement. Gene expression changes were analyzed through paired differential expression analysis and confirmed in a validation cohort. Weighted gene coexpression network analysis (WGCNA) was applied to identify thyroid state-related networks. Results: We detected 486 differentially expressed genes (fold-change >1.5; multiple testing corrected P value < 0.05), of which 76% were positively and 24% were negatively regulated. Gene ontology (GO) enrichment analysis revealed that three biological processes were significantly overrepresented, of which the process translational elongation showed the highest fold enrichment (7.3-fold, P = 1.8 × 10-6). WGCNA analysis independently identified various gene clusters that correlated with thyroid state. Further GO analysis suggested that thyroid state affects platelet function. Conclusions: Changes in thyroid state regulate numerous genes in human whole blood, predominantly TRα-expressing leukocytes. In addition, TH may regulate gene transcripts in platelets.

Genetic screening of regulatory regions of pituitary transcription factors in patients with idiopathic pituitary hormone deficiencies.

PURPOSE: Mutation frequencies of PROP1, POU1F1 and HESX1 in patients with combined pituitary hormone deficiencies (CPHD) vary substantially between populations. They are low in sporadic CPHD patients in Western Europe. However, most clinicians still routinely send DNA of their CPHD patients for genetic screening of these pituitary transcription factors. Before we can recommend against screening of PROP1, POU1F1 and HESX1 as part of routine work-up for Western-European sporadic CPHD patients, it is crucial to rule out possible defects in regulatory regions of these genes, which could also disturb the complex process of pituitary organogenesis. METHODS: The regulatory regions of PROP1, POU1F1 and HESX1 are not covered by Whole Exome Sequencing as they are largely located outside the coding regions. Therefore, we manually sequenced the regulatory regions, previously defined in the literature, of PROP1, POU1F1 and HESX1 among 88 Dutch patients with CPHD. We studied promoter SNPs in relation to phenotypic data. RESULTS: We found six known SNPs in the PROP1 promoter. In the POU1F1 promoter, we found one new variant and two known SNPs. We did not find any variant in the HESX1 promoter. CONCLUSION: Although the new POU1F1 variant might explain the phenotype of one patient, the general conclusion of this study is that variants in regulatory regions of PROP1, POU1F1 and HESX1 are rare in patients with sporadic CPHD in the Netherlands. We recommend that genetic screening of these pituitary transcription factors should no longer be part of routine work-up for Western-European, and especially Dutch, sporadic CPHD patients.

Outward-Open Model of Thyroid Hormone Transporter Monocarboxylate Transporter 8 Provides Novel Structural and Functional Insights.

Monocarboxylate transporter 8 (MCT8) facilitates cellular uptake and efflux of thyroid hormone (TH). Mutations in MCT8 result in severe intellectual and motor disability known as the Allan-Herndon-Dudley syndrome (AHDS). Previous studies have provided valuable insights into the putative mechanism of substrate binding in the inward-open conformation, required for TH efflux. The current study aims to delineate the mechanism of substrate binding in the outward-open conformation, required for TH uptake. Extensive chemical modification and site-directed mutagenesis studies were used to guide protein homology modeling of MCT8 in the outward-open conformation. Arg271 and Arg445 were modified by phenylglyoxal, which was partially prevented in the presence of substrate. Substrate docking in our outward-open model suggested an important role for His192 and Arg445 in substrate binding. Interestingly, mutations affecting these residues have been identified in patients who have AHDS. In addition, our outward-open model predicted the location of Phe189, Met227, Phe279, Gly282, Phe287, and Phe501 at the substrate-binding center, and their Ala substitution differentially affected the apparent Vmax and Km of T3 transport, with F189A, F279A, and F287A showing the highest impact. Thus, here we present an MCT8 homology model in the outward-open conformation, which supports the important role of His192 and Arg445 in substrate docking and identifies critical residues at the putative substrate-binding center. Our findings provide insights into MCT8 structure and function, which add to our understanding of the pathogenic mechanism of mutations found in patients who have AHDS and can be used to screen for novel substrates and inhibitors.

Thyroid Function and Premature Delivery in TPO Antibody-Negative Women: The Added Value of hCG.

Context: Human chorionic gonadotropin (hCG) stimulates thyroid function during pregnancy. We recently showed that thyroid autoimmunity severely attenuated the thyroidal response to hCG stimulation and that this may underlie the higher risk of premature delivery in thyroperoxidase antibody (TPOAb)-positive women. We hypothesized that a lower thyroidal response to hCG stimulation in TPOAb-negative women is also associated with a higher risk of premature delivery and preterm premature rupture of membranes (pPROM). Design, Setting, and Participants: Thyrotropin (TSH), free thyroxine (FT4), and hCG concentrations were available in 5644 TPOAb-negative women from a prospective cohort. We tested for interaction between TSH or FT4 and hCG in linear regression models for duration of pregnancy and logistic regression models for premature delivery/pPROM. Accordingly, analyses were stratified per TSH percentile (TSH ≥ 85th percentile) and hCG per 10,000 IU/L. Results: Women with high TSH and low hCG concentrations did not have a higher risk of premature delivery or pPROM, with protective effect estimates. In contrast, women with a high TSH concentration despite a high hCG concentration had twofold to 10-fold higher risk of premature delivery (Pdifference = 0.022) and an up to fourfold higher risk of pPROM (Pdifference = 0.079). hCG concentrations were not associated with premature delivery or pPROM. Conclusion: In TPOAb-negative women with high-normal TSH concentrations, only women with high hCG concentrations had a higher risk of premature delivery or pPROM. These results suggest a lower thyroidal response to hCG stimulation is also associated with premature delivery in TPOAb-negative women and that an additional measurement of hCG may improve thyroid-related risk assessments during pregnancy.