Cellular mechanisms of thyroid hormone homeostasis

Abstract The title of this thesis covers the whole world of local thyroid hormone (TH) transport, metabolism and action. Normal TH homeostasis is dependent on the intracellular availability of TH, as the genomic actions of TH are mediated by nuclear TH receptors (TRs) bound to promoter elements of TH responsive genes. The intracellular availability of TH, is dependent on adequate function of transporter proteins and deiodinating enzymes that convert T4 to the active form T3, as well as degradation of T3. Although we know that all described key players are important for normal TH homeostasis, many issues are still unresolved. The work in this thesis presents studies in which key players of TH homeostasis, in health and disease, are investigated. First, we discussed the impact of genetic variation in the type 2 deiodinase (D2). Next, new insights in the TH transport characteristics of the L-type amino acid transporters (LAT) 1-5 and monocarboxylate transporters (MCT) 8 and 10 were described. After that we discussed the ability of T4 to stimulate transcription of TH responsive genes and we explored gender differences in the age-related suppression of TH signalling. Finally, the associations between TH and trace elements were discussed

Tissue-specific suppression of thyroid hormone signaling in various mouse models of aging

DNA damage contributes to the process of aging, as underscored by premature aging syndromes caused by defective DNA repair. Thyroid state changes during aging, but underlying mechanisms remain elusive. Since thyroid hormone (TH) is a key regulator of metabolism, changes in TH signaling have widespread effects. Here, we reveal a significant common transcriptomic signature in livers from hypothyroid mice, DNA repair-deficient mice with severe (Csbm/m/Xpa-/-) or intermediate (Ercc1-/Δ-7) progeria and naturally aged mice. A strong induction of TH-inactivating deiodinase D3 and decrease of TH-activating D1 activities are observed in Csbm/m/Xpa-/- livers. Similar findings are noticed in Ercc1-/Δ-7, in naturally aged animals and in wild-type mice exposed to a chronic subtoxic dose of DNAdamaging agents. In contrast, TH signaling in muscle, heart and brain appears unaltered. These data show a strong suppression of TH signaling in specific peripheral organs in premature and normal aging, probably lowering metabolism, while other tissues appear to preserve metabolism. D3-mediated TH inactivation is unexpected, given its expression mainly in fetal tissues. Our studies highlight the importance of DNA damage as the underlying mechanism of changes in thyroid state. Tissue-specific regulation of deiodinase activities, ensuring diminished TH signaling, may contribute importantly to the protective metabolic response in aging

Association of antiepileptic drug usage, trace elements and thyroid hormone status

Background: Levels of thyroid hormone (TH) and trace elements (copper (Cu) and selenium (Se)) are important for development and function of the brain. Ant

Transport of iodothyronines by human l-type amino acid transporters

Thyroid hormone (TH) transporters facilitate cellular TH influx and efflux, which is paramount for normal physiology. The L-type amino acid transporters LAT1 and LAT2 are known to facilitate TH transport. However, the role of LAT3, LAT4, and LAT5 is still unclear. Therefore, the aim of this study was to further characterize TH transport by LAT1 and LAT2 and to explore possible TH transport by LAT3, LAT4, and LAT5. FLAG-LAT1-5 constructs were transiently expressed in COS1 cells. LAT1 and LAT2 were cotransfected with the CD98 heavy chain. Cellular transport was measured using 10 nM 125I-labeled T4, T3, rT3, 3,3'-T2, and 10 μM [125I]3μ-iodotyrosine (MIT) as substrates. Intracellular metabolism of these substrates was determined in cells cotransfected with either of the LATs with type 1 or type 3 deiodinase. LAT1 facilitated cellular uptake of all substrates and LAT2 showed a net uptake of T3, 3,3μ-T2, and MIT. Expression of LAT3 or LAT4 did not affect transport of T4 and T3 but resulted in the decreased cellular accumulation of 3,3μ-T2 and MIT. LAT5 did not facilitate the transport of any substrate. Cotransfection with LAT3 or LAT4 strongly diminished the cellular accumulation of 3,3μ-T2 and MIT by LAT1 and LAT2. These data were confirmed by metabolism studies. LAT1 and LAT2 show distinct preferences for the uptake of the different iodocompounds, whereas LAT3 and LAT4 specifically facilitate the 3,3μ-T2 and MIT efflux. Together our findings suggest that different sets of transporters with specific influx or efflux capacities may cooperate to regulate the cellular thyroid state

Functional and Medical Outcomes After Tailored Surgery for Pain Due to Chronic Pancreatitis

Objective: We measured a comprehensive set of outcome measures after different surgical procedures for painful chronic pancreatitis (CP) at long-term follow-up. Background: Pain caused by CP can be alleviated through operative intervention with type of procedure depending on anatomical abnormalities. Outcome measures include functional (pain relief, quality of life [QoL]), medical (endo- and exocrine function), and clinical (reoperation) results reported by patient. Methods: A cross-sectional cohort of 223 consecutive patients who underwent surgical drainage, head resection, or left-sided pancreas resection, depending on anatomical abnormalities, was analyzed. Participating patients were reassessed during a prospectively scheduled outpatient clinic visit. Results: At follow-up, 44 patients had died; 146 of 179 living patients consented to participate in the study. After 63 months (range: 14-268), 68% reported no or little pain, 19% reported intermediate pain, and 12% reported severe pain. Preoperative daily opioid use (OR: 3.04; 95% confidence interval [CI]: 1.09-8.49) and high numbers of preceding endoscopic procedures (OR [odds ratio]: 3.89; 95% CI: 1.01-14.9) were associated with persistent severe pain. Compared with the general population, physical more than mental QoL remained impaired (P <0.05). At follow-up, endocrine insufficiency was present in 57% of patients and exocrine insufficiency was present in 77%. Independently, a head resection and a reoperation for any cause were moderately associated with new-onset diabetes (P <0.1). Compared with patients who underwent left-sided resection, the risk of developing exocrine insufficiency after surgery was higher after drainage or head resection. After 20 months (interquartile range: 10-51) after surgery, 26 (12%) of 223 patients underwent 1 or more elective reoperations. Conclusions: Operative intervention for painful CP, tailored to anatomical abnormalities, results in excellent to fair long-term pain relief, but approximately 10% of patients do not respond. QoL scores remained slightly compromised. High preoperative pain levels, suggested through daily opioid use and high numbers of endoscopic procedures, are associated with less favorable outcom

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

Prevalent polymorphism in thyroid hormone-activating enzyme leaves a genetic fingerprint that underlies associated clinical syndromes

Context: A common polymorphism in the gene encoding the activating deiodinase (Thr92Ala-D2) is known to be associated with quality of life in millions of patients with hypothyroidism and with several organ-specific conditions. This polymorphism results in a single amino acid change within the D2 molecule where its susceptibility to ubiquitination and proteasomal degradation is regulated. Objective: To define the molecular mechanisms underlying associated conditions in carriers of the Thr92Ala-D2 polymorphism. Design, Setting, Patients: Microarray analyses of 19 postmortem human cerebral cortex samples were performed to establish a foundation for molecular studies via a cell model of HEK-293 cells stably expressing Thr92 or Ala92 D2. Results: The cerebral cortex of Thr92Ala-D2 carriers exhibits a transcriptional fingerprint that includes sets of genes involved in CNS diseases, ubiquitin,mitochondrial dysfunction (chromosomal genes encoding mitochondrial proteins), inflammation, apoptosis, DNA repair, and growth factor signaling. Similar findings were made in Ala92-D2-expressing HEK-293 cells and in both cases there was no evidence that thyroid hormone signaling was affected ie, the expression level of T3-responsive genes was unchanged, but that several other genes were differentially regulated. The combined microarray analyses (brain/cells) led to the development of an 81-gene classifier that correctly predicts the genotype of homozygous brain samples. In contrast to Thr92-D2, Ala92-D2 exhibits longer half-life and was consistently found in the Golgi. A number of Golgi-related genes were down-regulated in Ala92-D2-expressing cells, but were normalized after 24-h-treatment with the antioxidant N-acetylecysteine. Conclusions: Ala92-D2 accumulates in the Golgi, where its presence and/or ensuing oxidative stress disrupts basic cellular functions and increases pre-apoptosis. These findings are reminiscent to disease mechanisms observed in other neurodegenerative disorders such as Huntington’s disease, and could contribute to the unresolved neurocognitive symptoms of affected carriers

Functional analysis of novel genetic variation in the thyroid hormone activating type 2 deiodinase

Context: Thyroid hormones (TH) are important for normal brain development and abnormal TH regulation in the brain results in neurocognitive impairments. The type 2 deiodinase (D2) is important for local TH control in the brain by generating the active hormone T3 from its precursor T4. Dysfunction of D2 likely results in a neurocognitive phenotype. No mutations in D2 have been reported yet. Objective: The objective of the study was to identify D2 mutations in patients with intellectual disability and to test their functional consequences. Design, Setting, and Patients: The patients were selected from the multicenter Thyroid Origin of Psychomotor Retardation study, which is a cohort of 946 subjects with unexplained intellectual disability. Based on characteristic serum TH values, the coding region of the DIO2 gene was sequenced in 387 patients. Functional consequences were assessed by in vitro D2 assays or intact cell metabolism studies using cells transfected with wild-type or mutant D2. Results: Sequence analysis revealed two heterozygousmutations: c.11TA (p.L4H) in three subjects and c.305CT (p.T102I) in one subject. Sequence analysis of family members revealed several carriers, but no segregation was observed with thyroid parameters or neurocognitive phenotype. Extensive tests with different in vitro D2 assays did not show differences between wild-type and mutant D2. Conclusion: This study describes the identification and functional consequences of novel genetic variation in TH activating enzyme D2. Family studies and functional tests suggest that these variants do not underlie the neurocognitiveimpairment. Altogether our data provide evidence of the existence of rare but apparently harmless genetic variants of D2

Tissue-Specific Suppression of Thyroid Hormone Signaling in Various Mouse Models of Aging

DNA damage contributes to the process of aging, as underscored by premature aging syndromes caused by defective DNA repair. Thyroid state changes during aging, but underlying mechanisms remain elusive. Since thyroid hormone (TH) is a key regulator of metabolism, changes in TH signaling have widespread effects. Here, we reveal a significant common transcriptomic signature in livers from hypothyroid mice, DNA repair-deficient mice with severe (Csbm/m/Xpa-/-) or intermediate (Ercc1-/Δ-7) progeria and naturally aged mice. A strong induction of TH-inactivating deiodinase D3 and decrease of TH-activating D1 activities are observed in Csbm/m/Xpa-/- livers. Similar findings are noticed in Ercc1-/Δ-7, in naturally aged animals and in wild-type mice exposed to a chronic subtoxic dose of DNA-damaging agents. In contrast, TH signaling in muscle, heart and brain appears unaltered. These data show a strong suppression of TH signaling in specific peripheral organs in premature and normal aging, probably lowering metabolism, while other tissues appear to preserve metabolism. D3-mediated TH inactivation is unexpected, given its expression mainly in fetal tissues. Our studies highlight the importance of DNA damage as the underlying mechanism of changes in thyroid state. Tissue-specific regulation of deiodinase activities, ensuring diminished TH signaling, may contribute importantly to the protective metabolic response in aging.status: publishe