P R Larsen

Type 2 iodothyronine deiodinase is highly expressed in human thyroid.

Type 2 iodothyronine deiodinase (D2) is a recently cloned selenodeiodinase thought to provide intracellular 3,5,3 triiodothyronine (T3) to a restricted group of tissues. We report here the presence of D2 mRNA in human thyroid at levels 50-150-fold higher than in placenta. Surprisingly, while type 1 deiodinase (D1) is known to be present in human thyroid, D2 has not been evaluated previously. D2 mRNA was especially high in thyroids from Graves patients and in follicular adenomas. Stimulated thyroids had higher D2 to D1 mRNA ratios than normal or multinodular glands suggesting differential regulation of D1 and D2 expression. Microsomes from normal, Graves, and TSH-stimulated thyroids contained low Km D2 activity resistant to propylthiouracil (1 mM) or to inactivation by N-bromoacetyl T3, agents which block or inactivate D1. At 2 nM thyroxine (T4), 100 times the physiological-free T4 levels, 60-80% of T4 to T3 conversion in stimulated, but only 27% of that in normal thyroids, is catalyzed by D2. We conclude that intrathyroidal T4 to T3 conversion by D2 may contribute significantly to the relative increase in thyroidal T3 production in patients with Graves disease, toxic adenomas, and, perhaps, iodine deficiency.

A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene.

We identified two thyroid hormone response elements (TREs) in the 2.5-kb, 5-flanking region of the human gene encoding type 1 iodothyronine deiodinase (hdio1), an enzyme which catalyses the activation of thyroxine to 3,5,3-triiodothyronine (T3). Both TREs contribute equally to T3 induction of the homologous promoter in transient expression assays. The proximal TRE (TRE1), which is located at bp -100, has an unusual structure, a direct repeat of the octamer YYRGGTCA hexamer that is spaced by 10 bp. The pyrimidines in the -2 position relative to the core hexamer are both essential to function. In vitro binding studies of TRE1 showed no heterodimer formation with retinoid X receptor (RXR) beta or JEG nuclear extracts (containing RXR alpha) and bacterially expressed chicken T3 receptor alpha 1 (TR alpha) can occupy both half-sites although the 3 half-site is dominant. T3 causes dissociation of TR alpha from the 5 half-site but increases binding to the 3 half-site. Binding of a second TR to TRE1 is minimally cooperative; however, no cooperativity was noted for a functional mutant in which the half-sites are separated by 15 bp, implying that TRs bind as independent monomers. Nonetheless, T3 still causes TR dissociation from the DR+15, indicating that dissociation occurs independently of TR-TR contact and that rebinding of a T3-TR complex to the 3 half-site occurs because of its slightly higher affinity. A distal TRE (TRE2) is found at bp -700 and is a direct repeat of a PuGGTCA hexamer spaced by 4 bp. It has typical TR homodimer and TR-RXR heterodimer binding properties. The TRE1 of hdio1 is the first example of a naturally occurring TRE consisting of two relatively independent octamer sequences which do not require the RXR family of proteins for function.

Qualitative and quantitative differences in the pathways of extrathyroidal triiodothyronine generation between euthyroid and hypothyroid rats.

Propylthiouracil (PTU) in maximally inhibitory doses for liver and kidney iodothyronine 5-deiodinase activity (5D-I), reduces extrathyroidal T4 to T3 conversion by only 60-70% in euthyroid rats. A second pathway of T4 to T3 conversion (5D-II) has been found in pituitary, central nervous system, and brown adipose tissue. 5D-II is insensitive to PTU and increases in hypothyroidism, whereas 5D-I decreases in hypothyroid rats. Thyroxine (T4) and triiodothyronine (T3) kinetics were assessed in euthyroid and thyroidectomized rats by noncompartmental analysis after injecting [125I]T4 and [131I]T3. Neither the volume of distribution nor the rate of fractional removal of plasma T4 was affected by the thyroid status, but the fractional removal rate of T3 was approximately 50% reduced in hypothyroid rats (P less than 0.001). Fractional T4 to T3 conversion was 22% in euthyroid and 26% in hypothyroid rats. In euthyroid rats, sufficient PTU to inhibit liver and kidney 5D-I greater than 90% reduced serum [125I]T3 after [125I]T4 (results given as percent dose per milliliter X 10(-3) +/- SEM): 4 h, control 16 +/- 2 vs. PTU 4 +/- 1, P less than 0.005, and 22 h, control 6.4 +/- 0.4 vs. PTU 3.6 +/- 0.7, P less than 0.025. In thyroidectomized rats, the same dose of PTU also inhibited 5D-I in liver and kidney, but had no effect on the generation of serum [125I]T3 from [125I]T4. Similarly, after 1 microgram T4/100 g bw was given to thyroidectomized rats, serum T3 (radioimmunoassay) increased by 0.30 +/- 0.6 ng/ml in controls and 0.31 +/- 0.09 ng/ml in PTU-treated rats. However, when the dose of T4 was increased to 2-10 micrograms/100 g bw, PTU pretreatment significantly reduced the increment in serum T3. T3 clearance was not affected by PTU in hypothyroid rats. The 5D-II in brain, pituitary, and brown adipose tissue was reduced to less than or equal to 60% of control by 30 micrograms/100 g bw reverse T3 (rT3), an effect that lasted for at least 3 h after rT3 had been cleared. In rT3-pretreated thyroidectomized rats, the generation of [125I]T3 from tracer [125I]T4 was reduced in the serum: 6 +/- 1 vs. 12 +/- 1 X 10(-3)% dose/ml, P less than 0.01, during this 3-h period. We conclude that virtually all the T3 produced from low doses of exogenous T4 given to hypothyroid rats is generated via a PTU-insensitive pathway, presumably catalyzed by the 5D-II. This is a consequence of the enhanced activity of this low Km enzyme together with the concomitant decrease in the hepatic and renal 5D-I characteristic of the hypothyroid state. The results indicate that in some circumstances, 5D-II activity may contribute to the extracellular, as well as intracellular, T3 pool.

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. Methodology/Principal Findings After 6-weeks of HFD feeding D2KO mice gained 5.6% more body weight and had 28% more adipose tissue. Oxygen consumption (V02) was not different between genotypes, but D2KO mice had an increased respiratory exchange ratio (RER), suggesting preferential use of carbohydrates. Consistent with this, serum free fatty acids and β-hydroxybutyrate were lower in D2KO mice on a HFD, while hepatic triglycerides were increased and glycogen content decreased. Neither genotype showed glucose intolerance, but D2KO mice had significantly higher insulin levels during GTT independent of diet. Accordingly, during ITT testing D2KO mice had a significantly reduced glucose uptake, consistent with insulin resistance. Gene expression levels in liver, muscle, and brown and white adipose tissue showed no differences that could account for the increased weight gain in D2KO mice. However, D2KO mice have higher PEPCK mRNA in liver suggesting increased gluconeogenesis, which could also contribute to their apparent insulin resistance. Conclusions/Significance We conclude that the loss of the Dio2 gene has significant metabolic consequences. D2KO mice gain more weight on a HFD, suggesting a role for D2 in protection from diet-induced obesity. Further, D2KO mice appear to have a greater reliance on carbohydrates as a fuel source, and limited ability to mobilize and to burn fat. This results in increased fat storage in adipose tissue, hepatic steatosis, and depletion of liver glycogen in spite of increased gluconeogenesis. D2KO mice are also less responsive to insulin, independent of diet-induced obesity.

Physiological role and regulation of iodothyronine deiodinases: A 2011 update

T4 is a prohormone secreted by the thyroid. T4 has a long half life in circulation and it is tightly regulated to remain constant in a variety of circumstances. However, the availability of iodothyronine selenodeiodinases allow both the initiation or the cessation of thyroid hormone action and can result in surprisingly acute changes in the intracellular concentration of the active hormone T3, in a tissue-specific and chronologically-determined fashion, in spite of the constant circulating levels of the prohormone. This fine-tuning of thyroid hormone signaling is becoming widely appreciated in the context of situations where the rapid modifications in intracellular T3 concentrations are necessary for developmental changes or tissue repair. Given the increasing availability of genetic models of deiodinase deficiency, new insights into the role of these important enzymes are being recognized. In this review, we have incorporated new information regarding the special role played by these enzymes into our current knowledge of thyroid physiology, emphasizing the clinical significance of these new insights.

In vivo genomic footprinting of thyroid hormone-responsive genes in pituitary tumor cell lines.

We studied the effects of thyroid hormone (T3) on nuclear protein-DNA interactions by using dimethyl sulfate (DMS) and DNase I ligation-mediated PCR footprinting. We examined an endogenous gene the growth hormone (GH) gene, and a stably transfected plasmid containing the chicken lysozyme silencer (F2) T3 response element (TRE) gene, F2-TRE-TK-CAT, both in pituitary tumor (GC) cells. The 235-1 cell line, which expresses prolactin (PRL) and Pit-1, but not the T3 receptor (TR) or GH, was used as a control. DMS and DNase I footprinting identified protected G residues in the Pit-1, Sp1, and Zn-15 binding sites of the GH gene in GC, but not in 235-1, cells. There was no specific protection of the tripartite GH TRE at -180 bp against either DMS or DNase I in the absence or presence of T3 in either cell line. However, T3 increased protection of the Pit-1 and Sp1 binding sites against DMS in GC cells. In GC cells stably transfected with a plasmid containing F2-TRE-TK-CAT or TRalpha, chloramphenicol acetyltransferase expression was T3 inducible and DMS footprinting revealed both F2 TRE TR-binding half sites in a pattern suggesting the binding of TR homodimers before and during T3 exposure. We conclude that the GH gene is accessible to specific nuclear proteins in GC, but not in 235-1, cells and that T3 enhances this interaction, although there is no evidence of TR binding to the low-affinity rat GH TRE. The presence of TR binding to the high-affinity F2 TRE before and during T3 exposure suggests that reversible interaction of T3 with DNA-bound TRs, rather than transient T3-TR contact with TREs, determines the level of T3-stimulated transcriptional activation.

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. Another event in this feedback regulation may be the thyroid hormone-mediated induction of the TRH-inactivating pyroglutamyl peptidase II (PPII) in the hypothalamic tanycytes. This study compared the chronology of the acute effects of T(3) or T(4) on TSH suppression, TRH mRNA in the hypothalamic paraventricular nucleus (PVN), and the induction of tanycyte PPII. In wild-type mice, T(3) or T(4) caused a 50% decrease in serum TSH in hypothyroid mice by 5u200a h. There was no change in TRH mRNA in PVN over this interval, but there was a significant increase in PPII mRNA in the tanycytes. In mice with genetic inactivation of the type 2 iodothyronine deiodinase, T(3) decreased serum TSH and increased PPII mRNA levels, while T(4)-treatment was ineffective. We conclude that the rapid suppression of TSH in the hypothyroid mouse by T(3) occurs prior to a decrease in TRH mRNA though TRH inactivation may be occurring in the median eminence through the rapid induction of tanycyte PPII. The effect of T(4), but not T(3), requires the type 2 iodothyronine deiodinase.

Type-2 Iodothyronine 5′Deiodinase (D2) in Skeletal Muscle of C57Bl/6 Mice. II. Evidence for a Role of D2 in the Hypermetabolism of Thyroid Hormone Receptor α-Deficient Mice

Mice with ablation of the Thra gene have cold intolerance due to an as yet undefined defect in the activation of brown adipose tissue (BAT) uncoupling protein (UCP). They develop an alternate form of facultative thermogenesis, activated at temperatures below thermoneutrality and associated with hypermetabolism and reduced sensitivity to diet-induced obesity. A consistent finding in Thra-0/0 mice is increased type-2 iodothyronine deiodinase (D2) mRNA in skeletal muscle and other tissues. With an improved assay to measure D2 activity, we show here that this enzyme activity is increased in proportion to the mRNA and as a function of the ambient cold. The activation is mediated by the sympathetic nervous system in Thra-0/0, as it is in wild-type genotype mice, but the sympathetic nervous system effect is greater in Thra-0/0 mice. Using D2-ablated mice (Dio2-/-), we reported elsewhere and show here that, in spite of sharing a severe deficiency in BAT thermogenesis with Thra-0/0 and UCP1-knockout mice, they do not have an increase in oxygen consumption, and they gain more weight than wild-type controls when fed a high-fat diet. UCP3 mRNA is highly responsive to thyroid hormone, and it is increased in Thra-0/0 mice, particularly when fed high-fat diets. We show here that muscle UCP3 mRNA in hypothyroid Thra-0/0 mice is responsive to small dose-short regimens of T(4), indicating a role for locally, D2-generated T(3). Lastly, we show that bile acids stimulate not only BAT but also muscle D2 activity, and this is associated with stimulation of muscle UCP3 mRNA expression provided T(4) is present. These observations strongly support the concept that enhanced D2 activity in Thra-0/0 plays a critical role in their alternate form of facultative thermogenesis, stimulating increased fat oxidation by increasing local T(3) generation in skeletal muscle.

Type-2 Iodothyronine 5′Deiodinase in Skeletal Muscle of C57Bl/6 Mice. I. Identity, Subcellular Localization, and Characterization

RT-PCR shows that mouse skeletal muscle contains type-2 iodothyronine deiodinase (D2) mRNA. However, the D2 activity has been hard to measure. Except for newborn mice, muscle homogenates have no detectable activity. However, we have reported D2 activity in mouse muscle microsomes. As the mRNA, activity is higher in slow- than in fast-twitch muscle. We addressed here the major problems in measuring D2 activity in muscle by: homogenizing muscle in high salt to improve yield of membranous structures; separating postmitochondrial supernatant between 38 and 50% sucrose, to eliminate lighter membranes lacking D2; washing these with 0.1 M Na(2)CO(3) to eliminate additional contaminating proteins; pretreating all buffers with Chelex, to eliminate catalytic metals; and eliminating the EDTA from the assay, as this can bind iron that enhances dithiothreitol oxidation and promotes peroxidation reactions. Maximum velocity of T(3) generation by postgradient microsomes from red muscles was approximately 1100 fmol/(h · mg) protein with a Michaelis-Menten constant for T(4) of 1.5 nM. D2-specific activity of Na(2)CO(3)-washed microsomes was 6-10 times higher. The enrichment in D2 activity increased in parallel with the capacity of microsomes to load (sarco/endoplasmic reticulum Ca(2+)-ATPase) and bind Ca(2+) (calsequestrin), indicating that D2 resides in the inner sarcoplasmic reticulum, close to the nuclei. The presence of D3 in the sarcolemma suggests that the most of D2-generated T(3) acts locally. Estimates from maximum velocity, Michaelis-Menten constant, and muscle T(4) content suggest that mouse red, type-1, aerobic mouse muscle fibers can generate physiologically relevant amounts of T(3) and, further, that muscle D2 plays an important role in thyroid hormone-dependent muscle thermogenesis.