The selective loss of the type 2 iodothyronine deiodinase in mouse thyrotrophs increases basal TSH but blunts the thyrotropin response to hypothyroidism.

The type 2 iodothyronine deiodinase (D2) is essential for feedback regulation of TSH by T4. We genetically inactivated in vivo D2 in thyrotrophs using a mouse model of Cga-driven cre recombinase. Pituitary D2 activity was reduced 90% in the Cga-cre D2 knockout (KO) mice compared with control Dio2(fl/fl) mice.

Inflammation-inducible type 2 deiodinase expression in the leptomeninges, choroid plexus, and at brain blood vessels in male rodents.

Thyroid hormone regulates immune functions and has antiinflammatory effects. In promoter assays, the thyroid hormone-activating enzyme, type 2 deiodinase (D2), is highly inducible by the inflammatory transcription factor nuclear factor-κ B (NF-κB), but it is unknown whether D2 is induced in a similar fashion in vivo during inflammation. We first reexamined the effect of bacterial lipopolysaccharide (LPS) on D2 expression and NF-κB activation in the rat and mouse brain using in situ hybridization.

Catalysis leads to posttranslational inactivation of the type 1 deiodinase and alters its conformation.

Previously, it was shown that the type 1 deiodinase (D1) is subject to substrate-dependent inactivation that is blocked by pretreatment with the inhibitor of D1 catalysis, propylthiouracil (PTU). Using HepG2 cells with endogenous D1 activity, we found that while considerable D1-mediated catalysis of reverse tri-iodothyronine (rT(3)) is observed in intact cells, there was a significant loss of D1 activity in sonicates assayed from the same cells in parallel. This rT(3)-mediated loss of D1 activity occurs despite no change in D1 mRNA levels and is blocked by PTU treatment, suggesting a requirement for catalysis.

Thyroxine-induced expression of pyroglutamyl peptidase II and inhibition of TSH release precedes suppression of TRH mRNA and requires type 2 deiodinase.

Suppression of TSH release from the hypothyroid thyrotrophs is one of the most rapid effects of 3,3',5'-triiodothyronine (T(3)) or thyroxine (T(4)). It is initiated within an hour, precedes the decrease in TSHβ mRNA inhibition and is blocked by inhibitors of mRNA or protein synthesis. TSH elevation in primary hypothyroidism requires both the loss of feedback inhibition by thyroid hormone in the thyrotrophs and the positive effects of TRH.

Type II iodothyronine deiodinase provides intracellular 3,5,3'-triiodothyronine to normal and regenerating mouse skeletal muscle.

The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T(3) under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T(4)-to-T(3) conversion increases during differentiation in C(2)C(12) myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this.

Mice with a targeted deletion of the type 2 deiodinase are insulin resistant and susceptible to diet induced obesity.

Background: The type 2 iodothyronine deiodinase (D2) converts the pro-hormone thyroxine into T3 within target tissues. D2 is essential for a full thermogenic response of brown adipose tissue (BAT), and mice with a disrupted Dio2 gene (D2KO) have an impaired response to cold. BAT is also activated by overfeeding.

Type 2 iodothyronine deiodinase levels are higher in slow-twitch than fast-twitch mouse skeletal muscle and are increased in hypothyroidism.

Because of its large mass, relatively high metabolic activity and responsiveness to thyroid hormone, skeletal muscle contributes significantly to energy expenditure. Despite the presence of mRNA encoding the type 2 iodothyronine-deiodinase (D2), an enzyme that activates T(4) to T3, very low or undetectable activity has been reported in muscle homogenates of adult humans and mice. With a modified D2 assay, using microsomal protein, overnight incubation and protein from D2 knockout mouse muscle as a tissue-specific blank, we examined slow- and fast-twitch mouse skeletal muscles for D2 activity and its response to physiological stimuli.

Substitution of serine for proline in the active center of type 2 iodothyronine deiodinase substantially alters its in vitro biochemical properties with dithiothreitol but not its function in intact cells.

T(4) must be activated by its monodeiodination to T(3) by type 1 or 2 iodothyronine deiodinase (D1 and D2). Recent studies show that despite an approximately 2000-fold higher Michaelis constant (K(m); T(4)) for D1 than for D2 using dithiothreitol (DTT) as cofactor, D1 expressed in intact cells produces T(3) at free T(4) concentrations many orders of magnitude below its K(m). To understand the factors regulating D1 and D2 catalysis in vivo, we studied a mutant D2 with a proline at position 135 of the active center of D2 replaced with a serine, as found in D1.

Knockdown of the type 3 iodothyronine deiodinase (D3) interacting protein peroxiredoxin 3 decreases D3-mediated deiodination in intact cells.

The type 3 iodothyronine deiodinase (D3) is the primary deiodinase that inactivates thyroid hormone. Immunoprecipitation of D3, followed by fluorescent two-dimensional difference gel electrophoresis and mass spectrometry, identified peroxiredoxin 3 (Prx3) as a D3-associated protein. This interaction was confirmed using reverse coimmunoprecipitation, in which pull-down of Prx3 resulted in D3 isolation, and by fluorescence resonance energy transfer between cyan fluorescent protein-D3 and yellow fluorescent protein-Prx3.

The E3 ubiquitin ligase TEB4 mediates degradation of type 2 iodothyronine deiodinase.

The endoplasmic reticulum resident thyroid hormone-activating type 2 deiodinase (D2) is inactivated by ubiquitination via the hedgehog-inducible WSB-1. Ubiquitinated D2 can then be subsequently taken up by the proteasomal system or be reactivated by USP-33/20-mediated deubiquitination. Given that heterologously expressed D2 accumulates in Saccharomyces cerevisiae lacking the E3 ligase Doa10, we tested whether the human Doa10 ortholog, TEB4, plays a role in D2 ubiquitination and degradation.

Thyroid hormone activation in vascular smooth muscle cells is negatively regulated by glucocorticoid.

Background: Type 2 iodothyronine deiodinase (D2) catalyzes the production of triiodothyronine from thyroxine. D2 is present in rat aorta media, and there is a circadian variation in the D2 expression. In rat aorta media, the D2 activity exhibited the maximal value at 1200 hour and low value between 1800 and 2400 hour.

Factors that influence adherence with disease-modifying therapy in MS.

Background: The complexity and cost of injection treatment can represent a formidable challenge for patients affected by a chronic illness, particularly those whose treatment is primarily preventative and only modestly effective on the more conspicuous symptomatic aspects of the disease process. The aim of this investigation was to identify which factors most influenced nonadherent behavior with the available disease-modifying injection therapies for multiple sclerosis (MS).

Methods: A multicenter, observational (three-wave) study using surveys was developed and administered to patients with MS through the World Wide Web.

The thyroid hormone-inactivating deiodinase functions as a homodimer.

The type 3 deiodinase (D3) inactivates thyroid hormone action by catalyzing tissue-specific inner ring deiodination, predominantly during embryonic development. D3 has gained much attention as a player in the euthyroid sick syndrome, given its robust reactivation during injury and/or illness. Whereas much of the structure biology of the deiodinases is derived from studies with D2, a dimeric endoplasmic reticulum obligatory activating deiodinase, little is known about the holostructure of the plasma membrane resident D3, the deiodinase capable of thyroid hormone inactivation.

Hypoxia-inducible factor induces local thyroid hormone inactivation during hypoxic-ischemic disease in rats.

Thyroid hormone is a critical determinant of cellular metabolism and differentiation. Precise tissue-specific regulation of the active ligand 3,5,3'-triiodothyronine (T3) is achieved by the sequential removal of iodine groups from the thyroid hormone molecule, with type 3 deiodinase (D3) comprising the major inactivating pathway that terminates the action of T3 and prevents activation of the prohormone thyroxine. Using cells endogenously expressing D3, we found that hypoxia induced expression of the D3 gene DIO3 by a hypoxia-inducible factor-dependent (HIF-dependent) pathway.

Activation of thyroid hormone is transcriptionally regulated by epidermal growth factor in human placenta-derived JEG3 cells.

Human type II deiodinase is a master regulator of thyroid hormone activation in several tissues. In placenta, type II deiodinase mRNA levels and enzymatic activity are elevated only during the first trimester of pregnancy and then progressively decline. During this early stage, mitogens such as epidermal growth factor (EGF) have been shown to promote the proliferation of the trophoblast by acting through multiple mechanisms.

Glutathione depletion in hippocampal cells increases levels of H and L ferritin and glutathione S-transferase mRNAs.

Glutathione plays an essential role in maintaining cellular redox balance, protecting cells from oxidative stress and detoxifying xenobiotic compounds. Glutathione depletion has been implicated in neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Cells of neuronal origin are acutely sensitive to glutathione depletion, providing an avenue for studying the mechanisms invoked for neuronal survival in response to oxidant challenge.

Ubiquitination-induced conformational change within the deiodinase dimer is a switch regulating enzyme activity.

Ubiquitination is a critical posttranslational regulator of protein stability and/or subcellular localization. Here we show that ubiquitination can also regulate proteins by transiently inactivating enzymatic function through conformational change in a dimeric enzyme, which can be reversed upon deubiquitination. Our model system is the thyroid hormone-activating type 2 deiodinase (D2), an endoplasmic reticulum-resident type 1 integral membrane enzyme.

The small polyphenolic molecule kaempferol increases cellular energy expenditure and thyroid hormone activation.

Disturbances in energy homeostasis can result in obesity and other metabolic diseases. Here we report a metabolic pathway present in normal human skeletal muscle myoblasts that is activated by the small polyphenolic molecule kaempferol (KPF). Treatment with KPF leads to an approximately 30% increase in skeletal myocyte oxygen consumption.

Efficient incorporation of multiple selenocysteines involves an inefficient decoding step serving as a potential translational checkpoint and ribosome bottleneck.

Selenocysteine is incorporated into proteins via "recoding" of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3' untranslated region, termed selenocysteine incorporation sequence (SECIS) elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS element, selenoprotein P genes encode multiple UGAs and two SECIS elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein.

Metabolic instability of type 2 deiodinase is transferable to stable proteins independently of subcellular localization.

Thyroid hormone activation is catalyzed by two deiodinases, D1 and D2. Whereas D1 is a stable plasma membrane protein, D2 is resident in the endoplasmic reticulum (ER) and has a 20-min half-life due to selective ubiquitination and proteasomal degradation. Here we have shown that stable retention explains D2 residency in the ER, a mechanism that is nevertheless over-ridden by fusion to the long-lived plasma membrane protein, sodium-iodine symporter.

Supramolecular complexes mediate selenocysteine incorporation in vivo.

Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA([Ser]Sec) and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA([Ser]Sec), termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization.

Nuclear assembly of UGA decoding complexes on selenoprotein mRNAs: a mechanism for eluding nonsense-mediated decay?

Recoding of UGA from a stop codon to selenocysteine poses a dilemma for the protein translation machinery. In eukaryotes, two factors that are crucial to this recoding process are the mRNA binding protein of the Sec insertion sequence, SBP2, and the specialized elongation factor, EFsec. We sought to determine the subcellular localization of these selenoprotein synthesis factors in mammalian cells and thus gain insight into how selenoprotein mRNAs might circumvent nonsense-mediated decay.

Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.

While bile acids (BAs) have long been known to be essential in dietary lipid absorption and cholesterol catabolism, in recent years an important role for BAs as signalling molecules has emerged. BAs activate mitogen-activated protein kinase pathways, are ligands for the G-protein-coupled receptor (GPCR) TGR5 and activate nuclear hormone receptors such as farnesoid X receptor alpha (FXR-alpha; NR1H4). FXR-alpha regulates the enterohepatic recycling and biosynthesis of BAs by controlling the expression of genes such as the short heterodimer partner (SHP; NR0B2) that inhibits the activity of other nuclear receptors.

Atypical expression of type 2 iodothyronine deiodinase in thyrotrophs explains the thyroxine-mediated pituitary thyrotropin feedback mechanism.

T(4), the main product of thyroid secretion, is a critical signal in plasma that mediates the TSH-negative feedback mechanism. As a prohormone, T(4) must be converted to T(3) to acquire biological activity; thus, type 2 iodothyronine deiodinase (D2) is expected to play a critical role in this feedback mechanism. However, the mechanistic details of this pathway are still missing because, counterintuitively, D2 activity is rapidly lost in the presence of T(4) by a ubiquitin-proteasomal mechanism.