Identification of Human TRIAC Transmembrane Transporters.

Background: 3,5,3'-Triiodothyroacetic acid (TRIAC) is a T3-receptor agonist pharmacologically used in patients to mitigate T3 resistance. It is additionally explored to treat some symptoms of patients with inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8, SLC16A2). MCT8 is expressed along the blood-brain barrier, on neurons, astrocytes, and oligodendrocytes. Hence, pathogenic variants in MCT8 limit the access of TH into and their functions within the brain. TRIAC was shown to enter the brain independently of MCT8 and to modulate expression of TH-dependent genes. The aim of the study was to identify transporters that facilitate TRIAC uptake into cells. Methods: We performed a whole-genome RNAi screen in HepG2 cells stably expressing a T3-receptor-dependent luciferase reporter gene. Validation of hits from the primary and confirmatory secondary screen involved a counter screen with siRNAs and compared the cellular response to TRIAC to the effect of T3, in order to exclude siRNAs targeting the gene expression machinery. MDCK1 cells were stably transfected with cDNA encoding C-terminally myc-tagged versions of the identified TRIAC-preferring transporters. Several individual clones were selected after immunocytochemical characterization for biochemical characterization of their 125I-TRIAC transport activities. Results: We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporter 7 (OAT7), a sodium-independent organic anion transporter expressed in the plasma membrane in brain, pituitary, liver, and other organs. Competition with the SLC22A9/OAT7 substrate estrone-3-sulfate reduced 125I-TRIAC uptake. SLC29A2 encodes the equilibrative nucleoside transporter 2 (ENT2), which is ubiquitously expressed, including pituitary and brain. Coincubation with the SLC29A2/ENT2 inhibitor nitrobenzyl-6-thioinosine reduced 125I-TRIAC uptake. Moreover, ABCD1, an ATP-dependent peroxisomal pump, was identified as a 125I-TRIAC exporter in transfected MDCK1 cells. Conclusions: Knowledge of TRIAC transporter expression patterns, also during brain development, may thus in the future help to interpret observations on TRIAC effects, as well as understand why TRIAC may not show a desirable effect on cells or organs not expressing appropriate transporters. The identification of ABCD1 highlights the sensitivity of our established screening assay, but it may not hold significant relevance for patients undergoing TRIAC treatment.

Low thyroxine serves as an upstream regulator of ecophysiological adaptations in Ansell's mole-rats.

Introduction: About 10% of all rodent species have evolved a subterranean way of life, although life in subterranean burrows is associated with harsh environmental conditions that would be lethal to most animals living above ground. Two key adaptations for survival in subterranean habitats are low resting metabolic rate (RMR) and core body temperature (Tb). However, the upstream regulation of these traits was unknown thus far. Previously, we have reported exceptionally low concentrations of the thyroid hormone (TH) thyroxine (T4), and peculiarities in TH regulating mechanisms in two African mole-rat species, the naked mole-rat and the Ansell's mole-rat. Methods: In the present study, we treated Ansell's mole-rats with T4 for four weeks and analyzed treatment effects on the tissue and whole organism level with focus on metabolism and thermoregulation. Results: We found RMR to be upregulated by T4 treatment but not to the extent that was expected based on serum T4 concentrations. Our data point towards an extraordinary capability of Ansell's mole-rats to effectively downregulate TH signaling at tissue level despite very high serum TH concentrations, which most likely explains the observed effects on RMR. On the other hand, body weight was decreased in T4-treated animals and Tb was upregulated by T4 treatment. Moreover, we found indications of the hypothalamus-pituitary-adrenal axis potentially influencing the treatment effects. Conclusion: Taken together, we provide the first experimental evidence that the low serum T4 concentrations of Ansell's mole-rats serve as an upstream regulator of low RMR and Tb. Thus, our study contributes to a better understanding of the ecophysiological evolution of the subterranean lifestyle in African mole-rats.

In vitro and in vivo investigation of a thyroid hormone system-specific interaction with triazoles.

Alterations in thyroid hormones (TH) and thyroid-stimulating hormone levels are frequently found following exposure to chemicals of concern. Dysregulation of TH levels can severely perturb physiological growth, metabolism, differentiation, homeostasis in the adult and developmental processes in utero. A frequently identified mode of action for this interaction is the induction of hepatic detoxification mechanisms (e.g. SULTs and UGTs), which lead to TH conjugation and elimination and therefore interfere with hormonal homeostasis, fulfilling the endocrine disruptors (EDs) definition. A short-term study in rats with dietary exposure to cyproconazole, epoxiconazole and prochloraz was conducted and hepatocyte hypertrophy, hepatic UGT activity and Phase 1/2 gene expression inductions were observed together with changes in TH levels and thyroid follicular hypertrophy and hyperplasia. To test for specific interaction with the thyroid hormone system, in vitro assays were conducted covering thyroidal I-uptake (NIS), TH transmembranal transport via MCT8 and thyroid peroxidase (TPO) function. Assays for iodothyronine deiodinases (DIO1-DIO3) and iodotyrosine deiodinase (DEHAL1) were included, and from the animal experiment, Dio1 and Dehal1 activities were measured in kidney and liver as relevant local indicators and endpoints. The fungicides did not affect any TH-specific KEs, in vitro and in vivo, thereby suggesting hepatic conjugation as the dominant MoA.

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

AIMS: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. METHODS AND RESULTS: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. CONCLUSION: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.

Screening for endocrine disrupting chemicals inhibiting monocarboxylate 8 (MCT8) transporter facilitated thyroid hormone transport using a modified nonradioactive assay.

Early neurodevelopmental processes are strictly dependent on spatial and temporally modulated of thyroid hormone (TH) availability and action. Thyroid hormone transmembrane transporters (THTMT) are critical for regulating the local concentrations of TH, namely thyroxine (T4) and 3,5,3'-tri-iodothyronine (T3), in the brain. Monocarboxylate transporter 8 (MCT8) is one of the most prominent THTMT. Genetically induced deficiencies in expression, function or localization of MCT8 are associated with irreversible and severe neurodevelopmental adversities. Due to the importance of MCT8 in brain development, studies addressing chemical interferences of MCT8 facilitated T3 uptake are a crucial step to identify TH system disrupting chemicals with this specific mode of action. Recently a non-radioactive in vitro assay has been developed to rapidly screen for endocrine disrupting chemicals (EDCs) acting upon MCT8 mediated transport. This study explored the use of an UV-light digestion step as an alternative for the original ammonium persulfate (APS) digestion step. The non-radioactive TH uptake assay, with the incorporated UV-light digestion step of TH, was then used to screen a set of 31 reference chemicals and environmentally relevant substances to detect inhibition of MCT8-depending T3 uptake. This alternative assay identified three novel MCT8 inhibitors: methylmercury, bisphenol-AF and bisphenol-Z and confirmed previously known MCT8 inhibitors.

A microphysiological system for studying human bone biology under simultaneous control of oxygen tension and mechanical loading.

Throughout life, continuous remodelling is part of human bone biology and depends on the simultaneous action of physicochemical parameters such as oxygen tension and varying mechanical load. Thus, suitable model systems are needed, which allow concomitant modulation of these factors to recapitulate in vivo bone formation. Here, we report on the development of a first microphysiological system (MPS) that enables perfusion, environment-independent regulation of the oxygen tension as well as precise quantification and control of mechanical load. To demonstrate the use of the MPS for future studies on the (patho-)biology of bone, we built a simplified 3D model for early de novo bone formation. Primary human osteoblasts (OBs), which are the key players during this process, were seeded onto type I collagen scaffolds and cultured in the MPS. We could not only monitor cell viability and metabolism of OBs under varied physicochemical conditions, but also visualise the mineralisation of the extracellular matrix. In summary, we present a MPS that uniquely combines the independent control of physicochemical parameters and allows investigation of their influence on bone biology. We consider our MPS highly valuable to gain deeper insights into (patho-)physiological processes of bone formation in the future.

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

Comparative analysis of thyroid hormone systems in rodents with subterranean lifestyle.

African mole-rats are subterranean rodents inhabiting underground burrows. This habitat entails risks of overheating, hypoxia, and scarce food availability. Consequently, many subterranean species have evolved low basal metabolism and low body temperature, but the regulation of these traits at the molecular level were unknown. Measurements of serum thyroid hormone (TH) concentrations in African mole-rats have revealed a unique TH phenotype, which deviates from the typical mammalian pattern. Since THs are major regulators of metabolic rate and body temperature, we further characterised the TH system of two African mole-rat species, the naked mole-rat (Heterocephalus glaber) and the Ansell's mole-rat (Fukomys anselli) at the molecular level in a comparative approach involving the house mouse (Mus musculus) as a well-studied laboratory model in TH research. Most intriguingly, both mole-rat species had low iodide levels in the thyroid and naked mole-rats showed signs of thyroid gland hyperplasia. However, contrary to expectations, we found several species-specific differences in the TH systems of both mole-rat species, although ultimately resulting in similar serum TH concentrations. These findings indicate a possible convergent adaptation. Thus, our study adds to our knowledge for understanding adaptations to the subterranean habitat.

High throughput drug screening identifies resveratrol as suppressor of hepatic SELENOP expression.

INTRODUCTION: Selenium (Se) is an essential trace element that exerts its effects mainly as the proteinogenic amino acid selenocysteine within a small set of selenoproteins. Among all family members, selenoprotein P (SELENOP) constitutes a particularly interesting protein as it serves as a biomarker and serum Se transporter from liver to privileged tissues. SELENOP expression is tightly regulated by dietary Se intake, inflammation, hypoxia and certain substances, but a systematic drug screening has hitherto not been performed. METHODS: A compound library of 1861 FDA approved clinically relevant drugs was systematically screened for interfering effects on SELENOP expression in HepG2 cells using a validated ELISA method. Dilution experiments were conducted to characterize dose-responses. A most potent SELENOP inhibitor was further characterized by RNA-seq analysis to assess effect-associated biochemical pathways. RESULTS: Applying a 2-fold change threshold, 236 modulators of SELENOP expression were identified. All initial hits were replicated as biological triplicates and analyzed for effects on cell viability. A set of 38 drugs suppressed SELENOP expression more than three-fold, among which were cancer drugs, immunosuppressants, anti-infectious drugs, nutritional supplements and others. Considering a 90% cell viability threshold, resveratrol, vidofludimus, and antimony potassium-tartrate were the most potent substances with suppressive effects on extracellular SELENOP concentrations. Resveratrol suppressed SELENOP levels dose-dependently in a concentration range from 0.8 µM to 50.0 µM, without affecting cell viability, along with strong effects on key genes controlling metabolic pathways and vesicle trafficking. CONCLUSION: The results highlight an unexpected direct effect of the plant stilbenoid resveratrol, known for its antioxidative and health-promoting effects, on the central Se transport protein. The suppressive effects on SELENOP may increase liver Se levels and intracellular selenoprotein expression, thereby conferring additional protection to hepatocytes at the expense of systemic Se transport. Further physiological effects from this interaction require analyses in vivo and by clinical studies.

Mixture Risk Assessment of Complex Real-Life Mixtures-The PANORAMIX Project.

Humans are involuntarily exposed to hundreds of chemicals that either contaminate our environment and food or are added intentionally to our daily products. These complex mixtures of chemicals may pose a risk to human health. One of the goals of the European Union's Green Deal and zero-pollution ambition for a toxic-free environment is to tackle the existent gaps in chemical mixture risk assessment by providing scientific grounds that support the implementation of adequate regulatory measures within the EU. We suggest dealing with this challenge by: (1) characterising 'real-life' chemical mixtures and determining to what extent they are transferred from the environment to humans via food and water, and from the mother to the foetus; (2) establishing a high-throughput whole-mixture-based in vitro strategy for screening of real-life complex mixtures of organic chemicals extracted from humans using integrated chemical profiling (suspect screening) together with effect-directed analysis; (3) evaluating which human blood levels of chemical mixtures might be of concern for children's development; and (4) developing a web-based, ready-to-use interface that integrates hazard and exposure data to enable component-based mixture risk estimation. These concepts form the basis of the Green Deal project PANORAMIX, whose ultimate goal is to progress mixture risk assessment of chemicals.

Impact of metabolic stress induced by diets, aging and fasting on tissue oxygen consumption.

OBJECTIVE: Alterations in mitochondrial function play an important role in the development of various diseases, such as obesity, insulin resistance, steatohepatitis, atherosclerosis and cancer. However, accurate assessment of mitochondrial respiration ex vivo is limited and remains highly challenging. Using our novel method, we measured mitochondrial oxygen consumption (OCR) and extracellular acidification rate (ECAR) of metabolically relevant tissues ex vivo to investigate the impact of different metabolic stressors on mitochondrial function. METHODS: Comparative analyses of OCR and ECAR were performed in tissue biopsies of young mice fed 12 weeks standard-control (STD), high-fat (HFD), high-sucrose (HSD), or western diet (WD), matured mice with HFD, and 2year-old mice aged on STD with and without fasting. RESULTS: While diets had only marginal effects on mitochondrial respiration, respiratory chain complexes II and IV were reduced in adipose tissue (AT). Moreover, matured HFD-fed mice showed a decreased hepatic metabolic flexibility and prolonged aging increased OCR in brown AT. Interestingly, fasting boosted pancreatic and hepatic OCR while decreasing weight of those organs. Furthermore, ECAR measurements in AT could indicate its lipolytic capacity. CONCLUSION: Using ex vivo tissue measurements, we could extensively analyze mitochondrial function of liver, AT, pancreas and heart revealing effects of metabolic stress, especially aging.

Tentative Application of a Streamlined Protocol to Determine Organ-Specific Regulations of Deiodinase 1 and Dehalogenase Activities as Readouts of the Hypothalamus-Pituitary-Thyroid-Periphery-Axis.

In animal studies, both in basic science and in toxicological assessment of potential endocrine disruptors, the state of the thyroid hormone (TH) axis is often described and defined exclusively by the concentrations of circulating THs and TSH. Although it is known that the local, organ-specific effects of THs are also substantially regulated by local mechanisms such as TH transmembrane transport and metabolism of TH by deiodinases, such endpoint parameters of the axis are rarely assessed in these experiments. Currently developed in vitro assays utilize the Sandell-Kolthoff reaction, a photometric method of iodide determination, to test the effect of chemicals on iodotyrosine and iodothyronine deiodinases. Furthermore, this technology offers the possibility to determine the iodine content of various sample types (e.g., urine, ex vivo tissue) in a simple way. Here, we measured deiodinase type 1 and iodotyrosine dehalogenase activity by means of the Sandell-Kolthoff reaction in ex vivo samples of hypo- and hyperthyroid mice of two age groups (young; 3 months and old; 20 months). In thyroid, liver and kidney, organ-specific regulation patterns emerged across both age groups, which, based on this pilot study, may serve as a starting point for a deeper characterization of the TH system in relevant studies in the future and support the development of Integrated Approach for Testing and Assessment (IATA).

Design and Characterization of a Fluorescent Reporter Enabling Live-cell Monitoring of MCT8 Expression.

The monocarboxylate transporter 8 (MCT8) is a specific thyroid hormone transporter and plays an essential role in fetal development. Inactivating mutations in the MCT8 encoding gene SLC16A2 (solute carrier family 16, member 2) lead to the Allan-Herndon-Dudley syndrome, a condition presenting with severe endocrinological and neurological phenotypes. However, the cellular distribution pattern and dynamic expression profile are still not well known for early human neural development. OBJECTIVE: Development and characterization of fluorescent MCT8 reporters that would permit live-cell monitoring of MCT8 protein expression in vitro in human induced pluripotent stem cell (hiPSC)-derived cell culture models. METHODS: A tetracysteine (TC) motif was introduced into the human MCT8 sequence at four different positions as binding sites for fluorescent biarsenical dyes. Human Embryonic Kidney 293 cells were transfected and stained with fluorescein-arsenical hairpin-binder (FlAsH). Counterstaining with specific MCT8 antibody was performed. Triiodothyronine (T3) uptake was indirectly measured with a T3 responsive luciferase-based reporter gene assay in Madin-Darby Canine Kidney 1 cells for functional characterization. RESULTS: FlAsH staining and antibody counterstaining of all four constructs showed cell membrane expression of all MCT8 constructs. The construct with the tag after the first start codon demonstrated comparable T3 uptake to the MCT8 wildtype. CONCLUSION: Our data indicate that introduction of a TC-tag directly after the first start codon generates a MCT8 reporter with suitable characteristics for live-cell monitoring of MCT8 expression. One promising future application will be generation of stable hiPSC MCT8 reporter lines to characterize MCT8 expression patterns during in vitro neuronal development.

Natural Autoimmunity to Selenoprotein P Impairs Selenium Transport in Hashimoto's Thyroiditis.

The essential trace element selenium (Se) is needed for the biosynthesis of selenocysteine-containing selenoproteins, including the secreted enzyme glutathione peroxidase 3 (GPX3) and the Se-transporter selenoprotein P (SELENOP). Both are found in blood and thyroid colloid, where they serve protective functions. Serum SELENOP derives mainly from hepatocytes, whereas the kidney contributes most serum GPX3. Studies using transgenic mice indicated that renal GPX3 biosynthesis depends on Se supply by hepatic SELENOP, which is produced in protein variants with varying Se contents. Low Se status is an established risk factor for autoimmune thyroid disease, and thyroid autoimmunity generates novel autoantigens. We hypothesized that natural autoantibodies to SELENOP are prevalent in thyroid patients, impair Se transport, and negatively affect GPX3 biosynthesis. Using a newly established quantitative immunoassay, SELENOP autoantibodies were particularly prevalent in Hashimoto's thyroiditis as compared with healthy control subjects (6.6% versus 0.3%). Serum samples rich in SELENOP autoantibodies displayed relatively high total Se and SELENOP concentrations in comparison with autoantibody-negative samples ([Se]; 85.3 vs. 77.1 µg/L, p = 0.0178, and [SELENOP]; 5.1 vs. 3.5 mg/L, p = 0.001), while GPX3 activity was low and correlated inversely to SELENOP autoantibody concentrations. In renal cells in culture, antibodies to SELENOP inhibited Se uptake. Our results indicate an impairment of SELENOP-dependent Se transport by natural SELENOP autoantibodies, suggesting that the characterization of health risk from Se deficiency may need to include autoimmunity to SELENOP as additional biomarker of Se status.

The Nutritional Supply of Iodine and Selenium Affects Thyroid Hormone Axis Related Endpoints in Mice.

Selenium and iodine are the two central trace elements for the homeostasis of thyroid hormones but additional trace elements such as iron, zinc, and copper are also involved. To compare the primary effects of inadequate intake of selenium and iodine on the thyroid gland, as well as the target organs of thyroid hormones such as liver and kidney, mice were subjected to an eight-week dietary intervention with low versus adequate selenium and iodine supply. Analysis of trace element levels in serum, liver, and kidney demonstrated a successful intervention. Markers of the selenium status were unaffected by the iodine supply. The thyroid gland was able to maintain serum thyroxine levels even under selenium-deficient conditions, despite reduced selenoprotein expression in liver and kidney, including deiodinase type 1. Thyroid hormone target genes responded to the altered selenium and iodine supply, whereas the iron, zinc, and copper homeostasis remained unaffected. There was a notable interaction between thyroid hormones and copper, which requires further clarification. Overall, the effects of an altered selenium and iodine supply were pronounced in thyroid hormone target tissues, but not in the thyroid gland.

Age-dependent response to T4 overtreatment and recovery on systemic and organ level.

Thyroid hormone (TH) metabolism and cellular TH action are influenced by ageing. To investigate the response to thyroxine (T4) overtreatment, a kinetic study was conducted in young and aged mice with chronic hyperthyroidism and hormone withdrawal. Five and 22 months old male mice were treated with T4 or PBS over 5 weeks, followed by observation for up to 12 days. Serial analysis was performed for thyroid function parameters, transcript levels of TH target genes, deiodinase type 1 (DIO1) activity as well as serum lipids at 12, 24, 72, 144, 216, and 288 h after cessation of T4 administration. Higher FT3 concentrations and higher renal DIO1 activities were noted in aged mice 12 h after T4 withdrawal and marked thyroid-stimulating hormone elevation was found in aged mice after 12 days compared to respective controls. A biphasic expression pattern occurred for TH target genes in all organs and a hypothyroid organ state was observed at the end of the study, despite normalization of TH serum concentrations after 72 h. In line with this, mirror-image kinetics were detected for serum cholesterol and triglycerides in aged and young mice. Recovery from TH overtreatment in mice involves short- and medium-term adaption of TH metabolism on systemic and organ levels. Increased renal DIO1 activity may contribute to higher T3 concentrations and prolonged thyrotoxicosis followed by hypothyroidism in an aged-mouse organism. Translation of these findings in the clinical setting seems warranted and may lead to better management of hyperthyroidism and prevention of T4 overtreatment in aged patients.

Natural Autoimmunity to the Thyroid Hormone Monocarboxylate Transporters MCT8 and MCT10.

The monocarboxylate transporters 8 (MCT8) and 10 (MCT10) are important for thyroid hormone (TH) uptake and signaling. Reduced TH activity is associated with impaired development, weight gain and discomfort. We hypothesized that autoantibodies (aAb) to MCT8 or MCT10 are prevalent in thyroid disease and obesity. Analytical tests for MCT8-aAb and MCT10-aAb were developed and characterized with commercial antiserum. Serum samples from healthy controls, thyroid patients and young overweight subjects were analyzed, and prevalence of the aAb was compared. MCT8-aAb were additionally tested for biological effects on thyroid hormone uptake in cell culture. Positive MCT8-aAb and MCT10-aAb were detected in all three clinical cohorts analyzed. MCT8-aAb were most prevalent in thyroid patients (11.9%) as compared to healthy controls (3.8%) and overweight adolescents (4.2%). MCT8-aAb positive serum reduced T4 uptake in cell culture in comparison to MCT8-aAb negative control serum. Prevalence of MCT10-aAb was highest in the group of thyroid patients as compared to healthy subjects or overweight adolescents (9.0% versus 4.5% and 6.3%, respectively). We conclude that MCT8 and MCT10 represent autoantigens in humans, and that MCT8-aAb may interfere with regular TH uptake and signaling. The increased prevalence of MCT8-aAb and MCT10-aAb in thyroid disease suggests that their presence may be of pathophysiological relevance. This hypothesis deserves an analysis in large prospective studies.

Fat-body brummer lipase determines survival and cardiac function during starvation in Drosophila melanogaster.

The cross talk between adipose tissue and the heart has an increasing importance for cardiac function under physiological and pathological conditions. This study characterizes the role of fat body lipolysis for cardiac function in Drosophila melanogaster. Perturbation of the function of the key lipolytic enzyme, brummer (bmm), an ortholog of the mammalian ATGL (adipose triglyceride lipase) exclusively in the fly's fat body, protected the heart against starvation-induced dysfunction. We further provide evidence that this protection is caused by the preservation of glycerolipid stores, resulting in a starvation-resistant maintenance of energy supply and adequate cardiac ATP synthesis. Finally, we suggest that alterations of lipolysis are tightly coupled to lipogenic processes, participating in the preservation of lipid energy substrates during starvation. Thus, we identified the inhibition of adipose tissue lipolysis and subsequent energy preservation as a protective mechanism against cardiac dysfunction during catabolic stress.

Copper interferes with selenoprotein synthesis and activity.

Selenium and copper are essential trace elements for humans, needed for the biosynthesis of enzymes contributing to redox homeostasis and redox-dependent signaling pathways. Selenium is incorporated as selenocysteine into the active site of redox-relevant selenoproteins including glutathione peroxidases (GPX) and thioredoxin reductases (TXNRD). Copper-dependent enzymes mediate electron transfer and other redox reactions. As selenoprotein expression can be modulated e.g. by H2O2, we tested the hypothesis that copper status affects selenoprotein expression. To this end, hepatocarcinoma HepG2 cells and mice were exposed to a variable copper and selenium supply in a physiologically relevant concentration range, and transcript and protein expression as well as GPX and TXNRD activities were compared. Copper suppressed selenoprotein mRNA levels of GPX1 and SELENOW, downregulated GPX and TXNRD activities and decreased UGA recoding efficiency in reporter cells. The interfering effects were successfully suppressed by applying the copper chelators bathocuproinedisulfonic acid or tetrathiomolybdate. In mice, a decreased copper supply moderately decreased the copper status and negatively affected hepatic TXNRD activity. We conclude that there is a hitherto unknown interrelationship between copper and selenium status, and that copper negatively affects selenoprotein expression and activity most probably via limiting UGA recoding. This interference may be of physiological relevance during aging, where a particular shift in the selenium to copper ratio has been reported. An increased concentration of copper in face of a downregulated selenoprotein expression may synergize and negatively affect the cellular redox homeostasis contributing to disease processes.

Re-visiting autoimmunity to sodium-iodide symporter and pendrin in thyroid disease.

OBJECTIVE: Iodide transport across thyrocytes constitutes a critical step for thyroid hormone biosynthesis, mediated mainly by the basolateral sodium-iodide-symporter (NIS (SLC5A5)) and the apical anion exchanger pendrin (PDS (SLC26A4)). Both transmembrane proteins have been described as autoantigens in thyroid disease, yet the reports on autoantibody (aAb) prevalence and diagnostic usefulness are conflicting. Reasons for the inconclusive findings may be small study groups and principle differences in the technologies used. DESIGN: We decided to re-evaluate this important issue by establishing novel non-radioactive tests using full-length antigens and comparable protocols, and analyzing a large cohort of thyroid patients (n = 323) and control samples (n = 400). METHODS: NIS and PDS were recombinantly expressed as fusion protein with firefly luciferase (Luc). Stably transfected HEK293 cells were used as reproducible source of the autoantigens. RESULTS: Recombinant NIS-Luc showed iodide transport activity, indicating successful expression and correct processing. Commercial antibodies yielded dose-dependent responses in the newly established assays. Reproducibility of assay signals from patient sera was verified with respect to linearity, stability and absence of matrix effects. Prevalence of PDS-aAb was similar in thyroid patients and controls (7.7% vs 5.0%). NIS-aAb were more prevalent in patients than controls (7.7% vs 1.8%), especially in Graves' Disease (12.3%). Neither NIS-aAb nor PDS-aAb concentrations were related to TPO-aAb or TSH-receptor-aAb concentrations, or to serum zinc or selenium status. CONCLUSIONS: Our data highlight a potential relevance of autoimmunity against NIS for thyroid disease, whereas an assessment of PDS-aAb in thyroid patients seems not to be of diagnostic value (yet).