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Type 2 iodothyronine deiodinase (D2) catalyzes the first step in thyroid hormone action, the deiodination of T4 to T3 . Endogenous D2 activity is posttranslationally regulated by substrate that accelerates its degradation through the ubiquitin-proteasome pathway. To understand how D2 activity correlates with D2 protein during its normal decay and rT3-induced down-regulation, HEK-293 cells, transiently expressing human D2, were labeled with Na75SeO3 and then treated with 100μ m cycloheximide (CX), 30 nm rT3, and/or 10 μm MG132, a specific proteasome inhibitor, for 2–4 h. D2 protein and enzyme activity changed in parallel, disappearing with a half-life of 2 h in the presence of CX, or 1 h when CX + rT3 were combined. Treatment with MG132 blocked these effects. We created selenocysteine (Sec) 133 to cysteine (Cys) or alanine (Ala) D2 mutants, without changing Sec 266. The CysD2 activity and protein levels were also parallel, with a similar half-life of approximately 2 h, whereas the rT3-induced D2 down-regul...

To investigate the role of type II 5′-deiodinase (5′D-II) in the expression of mitochondrial uncoupling protein (UCP) in brown adipose tissue (BAT), we injected intact male rats with reverse (r) 3,5,3′-triiodothyronine (T3; 100 μg ⋅ 100 g body wt−1⋅ day−1), an inhibitor of 5′D-II, for 2–5 days. UCP decreased by ∼20% in rats kept at 28°C and failed to increase during cold exposure (4°C). Next, thyroxine treatment (1–10 μg ⋅ 100 g body wt−1⋅ day−1) increased nuclear T3in rats kept at 28 or 4°C. In these rats, nuclear T3correlated positively with UCP. In addition, T3(1–50 μg ⋅ 100 g body wt−1⋅ day−1) given to intact rats (5–15 days; 28°C) induced an approximately twofold increase in UCP. In these T3-treated animals, the interscapular BAT thermal response to norepinephrine infusion also correlated positively with T3dose and UCP content. Treatment with propranolol or reserpine failed to block the T3induction of UCP (∼1.8- and ∼2.3-fold). The results emphasize the importance of local 5′D-II and reveal an independent role of T3in the expression of UCP.

To investigate the effects of thyroxine (T4) administration on bone mass, five 81-day-old female rats were treated with T4 (25 microg of T4/100 g of body weight [bw]/day), and bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) 28 days later. The BMD values for the total skeleton, femoral, and tibial subsegments were lower than in controls (por = 0.05). The lumbar spine (L2-L5) was not significantly affected by T4 treatment. Next, thirty-seven 211 +/- 1.5 (mean +/- SEM)-day-old female rats were divided into six groups as follows: (1) control; (2) ovariectomized (OVX); (3) 1xT4 (approximately 1.0 microg of T4/100 g of bw/day; approximately physiological replacement dose); (4) OVX + 1xT4; (5) 2xT4 (approximately 2.0 microg of T4/100 g of bw/day); (6) OVX + 2xT4. DXA scans were performed at days 0 and 85. Control rats showed a generalized BMD increase, as opposed to a decrease in OVX rats. The trabecular bone volume of the fifth lumbar vertebra was also lower in OVX rats than in controls (p0.05). The 1xT4 treatment had no effect on BMD of intact rats, while treatment with 2xT4 impaired the expected BMD increase. Unexpectedly, the OVX + 1xT4 group presented a generalized BMD increase that was significant for the total skeleton, L2-L5, and femoral subsegments (p0.05), comparable to controls. Treating OVX animals with 2xT4 did not potentiate the osteopenic effects of estrogen deficiency, nor did it reverse the osteopenic effects of OVX. In conclusion, treatment with high doses of T4 caused BMD to decrease substantially, particularly at the femur, whereas near physiological doses of T4 prevented bone loss associated with OVX, and regardless of bone type (trabecular or cortical), the skeleton site seems to be a more important determinant of the effects of thyroid hormone on bone mass.

By administration of bacterial lipopolysaccharide (LPS) to intact and T4-replaced thyroidectomized rats, we demonstrate that in contrast to the cortex and anterior pituitary, there is a persistent increase in type 2 iodothyronine deiodinase (D2) activity in the mediobasal hypothalamus (MBH). We propose that endotoxin-induced D2 activation in the MBH is independent of circulating levels of thyroid hormone and that this mechanism may contribute to central hypothyroidism associated with infection.

Intraventricular hemorrhage (IVH) remains a major cause of white matter injury in preterm infants with no viable therapeutic strategy to restore myelination. Maturation of oligodendrocytes and myelination is influenced by thyroid hormone (TH) signaling, which is mediated by TH receptor α (TRα) and TRβ. In the brain, cellular levels of TH are regulated by deiodinases, with deiodinase-2 mediating TH activation and deiodinase-3 TH inactivation. Therefore, we hypothesized that IVH would decrease TH signaling via changes in the expression of deiodinases and/or TRs, and normalization of TH signaling would enhance maturation of oligodendrocytes and myelination in preterm infants with IVH. These hypotheses were tested using both autopsy materials from human preterm infants and a rabbit model of IVH. We found that deiodinase-2 levels were reduced, whereas deiodinase-3 levels were increased in brain samples of both humans and rabbits with IVH compared with controls without IVH. TRα expression was also increased in human infants with IVH. Importantly, treatment with TH accelerated the proliferation and maturation of oligodendrocytes, increased transcription of Olig2 and Sox10 genes, augmented myelination, and restored neurological function in pups with IVH. Consistent with these findings, the density of myelinating oligodendrocytes was almost doubled in TH-treated human preterm infants compared with controls. Thus, in infants with IVH the combined elevation in deiodinase-3 and reduction in deiodinase-2 decreases TH signaling that can be worsened by an increase in unliganded TRα. Given that TH promotes neurological recovery in IVH, TH treatment might improve the neurodevelopmental outcome of preterm infants with IVH.

Cells are not passive bystanders in the process of hormonal signaling and instead can actively customize hormonal action. While diffusing from the plasma membrane to the nucleus, thyroid hormone is modified via the action of thioredoxin fold–containing selenoenzymes known as deiodinases. Whereas the type II deiodinase (D2) converts the prohormone thyroxine (T4) to the biologically active T3, the type III deiodinase (D3) converts it to reverse T3, an inactive metabolite. D3 also inactivates T3 to T2, terminating thyroid hormone action. Therefore, D2 provides cells with the ability to produce extra amounts of T3 and thus enhances thyroid hormone signaling. In contrast, expression of D3 results in the opposite action. In addition, the D2 protein is unique in that it can be switched off and on via an ubiquitin-regulated mechanism, triggered by catalysis of T4. Induction of D2 enhances local thyroid hormone signaling and energy expenditure during activation of brown adipose tissue by cold exposure or high fat diet. On the other hand, induction of D3 in myocardium and brain during ischemia and hypoxia decreases energy expenditure as part of a homeostatic mechanism to slow down cell metabolism in the face of limited O2 supply.

Altered glucose metabolism in the heart is an important characteristic of cardiovascular and metabolic disease. Because thyroid hormones have major effects on peripheral metabolism, we examined the metabolic effects of heart-selective increase in T3 using transgenic mice expressing human type 2 iodothyronine deiodinase (D2) under the control of the α-myosin heavy chain promoter (MHC-D2). Hyperinsulinemic-euglycemic clamps showed normal whole-body glucose disposal but increased hepatic insulin action in MHC-D2 mice as compared to wild-type (WT) littermates. Insulin-stimulated glucose uptake in heart was not altered, but basal myocardial glucose metabolism was increased by more than two-fold in MHC-D2 mice. Myocardial lipid levels were also elevated in MHC-D2 mice, suggesting an overall up-regulation of cardiac metabolism in these mice. The effects of doxorubicin (DOX) treatment on cardiac function and structure were examined using M-mode echocardiography. DOX treatment caused a significant reduction in ventricular fractional shortening and resulted in more than 50% death in WT mice. In contrast, MHC-D2 mice showed increased survival rate after DOX treatment, and this was associated with a six-fold increase in myocardial glucose metabolism and improved cardiac function. Myocardial activity and expression of AMPK, GLUT1, and Akt were also elevated in MHC-D2 and WT mice following DOX treatment. Thus, our findings indicate an important role of thyroid hormone in cardiac metabolism and further suggest a protective role of glucose utilization in DOX-mediated cardiac dysfunction.

FUNDAMENTO: Há grande controvérsia quanto ao diagnóstico de Insuficiência Renal Aguda (IRA), existindo mais de 30 diferentes definições. OBJETIVO: Avaliar a incidência e os fatores de risco para desenvolvimento de IRA no pós-operatório de cirurgia cardíaca de acordo com os critérios RIFLE, AKIN e KDIGO, e comparar o poder prognóstico desses critérios. MÉTODOS: Estudo de corte transversal que incluiu 321 pacientes (62 [53 - 71] anos, 140 homens) consecutivamente submetidos a cirurgia cardíaca entre junho de 2011 e janeiro de 2012. Os pacientes foram acompanhados por 30 dias, com vistas ao desenvolvimento de um desfecho composto (mortalidade, necessidade de diálise e internação prolongada). RESULTADOS: A incidência de IRA variou de 15% - 51%, conforme o critério diagnóstico adotado. Enquanto a idade se associou ao risco de IRA nos três critérios, houve variação nos demais determinantes. Durante o acompanhamento, 89 pacientes apresentaram o desfecho e todos os critérios se associaram ao risco aumentado na análise Cox univariada e após o ajuste para idade, sexo, diabetes e tipo de cirurgia. Contudo, após novo ajuste para tempo de circulação extracorpórea e presença de baixo débito cardíaco, apenas o diagnóstico de IRA pelo critério KDIGO manteve esta associação significativa (HR= 1,89 [95% IC: 1,18 - 3,06]). CONCLUSÕES: A incidência e os fatores de risco para IRA pós-cirurgia cardíaca têm grande variação de acordo com os critérios diagnósticos utilizados. Em nossa análise, o critério KDIGO se mostrou superior ao AKIN e ao RIFLE quanto ao seu poder prognóstico. BACKGROUND: There is considerable controversy regarding the diagnosis of Acute Kidney Injury (AKI), and there are over 30 different definitions. OBJECTIVE: To evaluate the incidence and risk factors for the development of AKI following cardiac surgery according to the RIFLE, AKIN and KDIGO criteria, and compare the prognostic power of these criteria. METHODS: Cross-sectional study that included 321 consecutively patients (median age 62 [53-71] years; 140 men) undergoing cardiac surgery between June 2011 and January 2012. The patients were followed for up to 30 days, for a composite outcome (mortality, need for dialysis and extended hospitalization). RESULTS: The incidence of AKI ranged from 15% - 51%, accordingly to the diagnostic criterion adopted. While age was associated with risk of AKI in the three criteria, there were variations in the remaining risk factors. During follow-up, 89 patients developed the outcome and all criteria were associated with increased risk in the univariate Cox analysis and after adjustment for age, gender, diabetes, and type of surgery. However, after further adjustment for extracorporeal circulation and the presence of low cardiac output, only AKI diagnosed by the KDIGO criterion maintained this significant association (HR= 1.89 [95% CI: 1.18 - 3.06]). CONCLUSION: The incidence and risk factors for AKI after cardiac surgery vary significantly according to the diagnostic criteria used. In our analysis, AKI the KDIGO criterion was superior to AKIN and RIFLE with regard its prognostic power.

A multicentre randomised phase III trial in chemotherapy-naive patients with advanced non-small-cell lung cancer (NSCLC) was undertaken to compare the therapeutic activity and toxicity of a cisplatin/carboplatin-etoposide-vinorelbine combination with that of a cisplatin-etoposide regimen. Patients with advanced (stage IIIB-IV) NSCLC were randomised, after stratification for stage (IIIB-IV) and performance status (0-1 and 2), to receive either (A) CDDP 40 mg m-2 + VP16 100 mg m-2 on days 1-3 as standard treatment or (B) CBDCA 250 mg m-2 on day 1 + CDDP 30 mg m-2 on days 2 and 3 + VP16 100 mg m-2 on days 1-3 + NVB 30 mg m-2 on day 1. Therapy was recycled on day 29 in both arms. We hypothesised a 15% minimum increment in the response rate with the experimental regimen over the 25% expected activity rate of the standard regimen. A two-stage design was chosen, which permitted the early termination of the trial (after the accrual of 52 patients in each arm) if the difference in response rates between the two regimens was less than 3% at the end of the first stage. A total of 112 patients (arm A = 57, arm B = 55) were enrolled in the study (53 with stage IIIB and 59 with stage IV), of which 105 eligible patients were evaluable for response on an "intention to treat' basis. Seven patients were excluded because they did not fulfil the inclusion criteria. Fifteen responses were observed in arm A (28%, 95% CI = 17-42) and 13 (one complete) in arm B (25%, 95% CI = 13-37). On multivariate logistic analysis, treatment did not affect the response rate, while stage IV and performance status 2 were significantly associated with a lower probability of response. Median survivals were similar in the two arms (31 vs 27 weeks). The experimental regimen was associated with an extremely poor median survival in patients with poor performance status (21 weeks). On Cox analysis, treatment failed to show a significant impact on survival: stage IV (relative risk = 1.6. CI = 1.0-2.6, P = 0.036) was the only prognostic variable significantly associated with a worse survival outcome and, although poor performance status adversely affected survival, this effect did not reach the level of statistical significance (relative risk = 1.6, CI = 0.98-2.5; P = 0.063). There were no significant differences in non-haematological toxicities between the two arms, although three patients in the control arm had to discontinue the treatment because of the persistence of severe nephrotoxicity (two patients) or neurotoxicity (one patient). In contrast, a significant increase in both neutropenia and thrombocytopenia was observed in the experimental arm. Four treatment-related deaths were registered in arm B (two due to neutropenic sepsis, one to myocardial failure and one to acute renal failure) compared with one toxic death (acute renal failure) in arm A. In view of these results, the trial was stopped and the null hypothesis (< 15% increase in response rate with the experimental regimen) has been accepted. Therefore, our combination does not deserve further evaluation as first-line treatment in advanced NSCLC patients. As our data suggest that an aggressive chemotherapy might have a negative impact on survival of patients with poor performance status, trials to evaluate the activity of new regimens should be conducted separately for each subset of patients with different performance status.

Thyroid hormone replacement has been used for more than a century to treat hypothyroidism. Natural thyroid preparations (thyroid extract, desiccated thyroid, or thyroglobulin), which contain both thyroxine (T4) and triiodothyronine (T3), were the first pharmacologic treatments available and dominated the market for the better part of the 20th century. Dosages were adjusted to resolve symptoms and to normalize the basal metabolic rate and/or serum protein-bound iodine level, but thyrotoxic adverse effects were not uncommon. Two major developments in the 1970s led to a transition in clinical practice: 1) The development of the serum thyroid-stimulating hormone (TSH) radioimmunoassay led to the discovery that many patients were overtreated, resulting in a dramatic reduction in thyroid hormone replacement dosage, and 2) the identification of peripheral deiodinase-mediated T4-to-T3 conversion provided a physiologic means to justify l-thyroxine monotherapy, obviating concerns about inconsistencies with desiccated thyroid. Thereafter, l-thyroxine mono-therapy at doses to normalize the serum TSH became the standard of care. Since then, a subgroup of thyroid hormone–treated patients with residual symptoms of hypothyroidism despite normalization of the serum TSH has been identified. This has brought into question the inability of l-thyroxine monotherapy to universally normalize serum T3 levels. New research suggests mechanisms for the inadequacies of l-thyroxine monotherapy and highlights the possible role for personalized medicine based on deiodinase polymorphisms. Understanding the historical events that affected clinical practice trends provides invaluable insight into formulation of an approach to help all patients achieve clinical and biochemical euthyroidism.

Background Thyroid hormone signaling is critical for development, growth and metabolic control in vertebrates. Although serum concentration of thyroid hormone is remarkable stable, deiodinases modulate thyroid hormone signaling on a time- and cell-specific fashion by controlling the activation and inactivation of thyroid hormone. Scope of the review This review covers the recent advances in D2 biology, a member of the iodothyronine deiodinase family, thioredoxin fold‐containing selenoenzymes that modify thyroid hormone signaling in a time- and cell-specific manner. Major conclusions D2-catalyzed T3 production increases thyroid hormone signaling whereas blocking D2 activity or disruption of the Dio2 gene leads to a state of localized hypothyroidism. D2 expression is regulated by different developmental, metabolic or environmental cues such as the hedgehog pathway, the adrenergic- and the TGR5-activated cAMP pathway, by xenobiotic molecules such as flavonols and by stress in the endoplasmic reticulum, which specifically reduces de novo synthesis of D2 via an eIF2a-mediated mechanism. Thus, D2 plays a central role in important physiological processes such as determining T3 content in developing tissues and in the adult brain, and promoting adaptive thermogenesis in brown adipose tissue. Notably, D2 is critical in the T4-mediated negative feed-back at the pituitary and hypothalamic levels, whereby T4 inhibits TSH and TRH expression, respectively. Notably, ubiquitination is a major step in the control of D2 activity, whereby T4 binding to and/or T4 catalysis triggers D2 inactivation by ubiquitination that is mediated by the E3 ubiquitin ligases WSB-1 and/or TEB4. Ubiquitinated D2 can be either targeted to proteasomal degradation or reactivated by deubiquitination, a process that is mediated by the deubiquitinases USP20/33 and is important in adaptive thermogenesis. General significance Here we review the recent advances in the understanding of D2 biology focusing on the mechanisms that regulate its expression and their biological significance in metabolically relevant tissues. This article is part of a Special Issue entitled Thyroid hormone signalling.

Brown adipose tissue (BAT) is predominantly regulated by the sympathetic nervous system (SNS) and the adrenergic receptor signaling pathway. Knowing that a mouse with triple β-receptor knockout (KO) is cold intolerant and obese, we evaluated the independent role played by the β1isoform in energy homeostasis. First, the 30 min i.v. infusion of norepinephrine (NE) or the β1selective agonist dobutamine (DB) resulted in similar interscapular BAT (iBAT) thermal response in WT mice. Secondly, mice with targeted disruption of the β1gene (KO of β1adrenergic receptor (β1KO)) developed hypothermia during cold exposure and exhibited decreased iBAT thermal response to NE or DB infusion. Thirdly, when placed on a high-fat diet (HFD; 40% fat) for 5 weeks, β1KO mice were more susceptible to obesity than WT controls and failed to develop diet-induced thermogenesis as assessed by BATUcp1mRNA levels and oxygen consumption. Furthermore, β1KO mice exhibited fasting hyperglycemia and more intense glucose intolerance, hypercholesterolemia, and hypertriglyceridemia when placed on the HFD, developing marked non-alcoholic steatohepatitis. In conclusion, the β1signaling pathway mediates most of the SNS stimulation of adaptive thermogenesis.

In neurons, the type 3 deiodinase (D3) inactivates thyroid hormone and reduces oxygen consumption, thus creating a state of cell-specific hypothyroidism. Here we show that hypoxia leads to nuclear import of D3 in neurons, without which thyroid hormone signaling and metabolism cannot be reduced. After unilateral hypoxia in the rat brain, D3 protein level is increased predominantly in the nucleus of the neurons in the pyramidal and granular ipsilateral layers, as well as in the hilus of the dentate gyrus of the hippocampal formation. In hippocampal neurons in culture as well as in a human neuroblastoma cell line (SK-N-AS), a 24 h hypoxia period redirects active D3 from the endoplasmic reticulum to the nucleus via the cochaperone Hsp40 pathway. Preventing nuclear D3 import by Hsp40 knockdown resulted an almost doubling in the thyroid hormone-dependent glycolytic rate and quadrupling the transcription of thyroid hormone target gene ENPP2. In contrast, Hsp40 overexpression increased nuclear import of D3 and minimized thyroid hormone effects in cell metabolism. In conclusion, ischemia/hypoxia induces an Hsp40-mediated translocation of D3 to the nucleus, facilitating thyroid hormone inactivation proximal to the thyroid hormone receptors. This adaptation decreases thyroid hormone signaling and may function to reduce ischemia-induced hypoxic brain damage.

Current guidelines recommend that hypothyroid patients should be treated with levothyroxine, which in the vast majority of the cases leads to resolution of the symptoms and normalization of serum free T4 (FT4), T3 and TSH levels. However, a small group of hypothyroid patients remain symptomatic for neurocognitive dysfunction despite normal serum FT4 and TSH, which could be explained by localized brain hypothyroidism. More than half of the T3 in the brain is produced locally via the action of the type II deiodinase (D2) and variability/defects in this pathway could explain the residual symptoms. If this rationale is correct, adding liothyronine to the replacement therapy could prove beneficial. However, with a few exceptions, several clinical trials failed to identify any beneficial effects of combined therapy. More recently, the results of a large clinical trial revealed a better neurocognitive outcome with combined therapy only in hypothyroid patients carrying a polymorphism in the DIO2 gene. This obviously needs to be confirmed by other groups but it is tempting to speculate that combined levothyroxine and liothyronine has a place in the treatment of hypothyroidism, for some.

Hypothalamic neurosecretory systems are fundamental regulatory circuits influenced by thyroid hormone. Monocarboxylate-transporter-8 (MCT8)-mediated uptake of thyroid hormone followed by type 3 deiodinase (D3)-catalyzed inactivation represent limiting regulatory factors of neuronal T3 availability. In the present study we addressed the localization and subcellular distribution of D3 and MCT8 in neurosecretory neurons and addressed D3 function in their axons. Intense D3-immunoreactivity was observed in axon varicosities in the external zone of the rat median eminence and the neurohaemal zone of the human infundibulum containing axon terminals of hypophysiotropic parvocellular neurons. Immuno-electronmicroscopy localized D3 to dense-core vesicles in hypophysiotropic axon varicosities. N-STORM-superresolution-microscopy detected the active center containing C-terminus of D3 at the outer surface of these organelles. Double-labeling immunofluorescent confocal microscopy revealed that D3 is present in the majority of GnRH, CRH and GHRH axons but only in a minority of TRH axons, while absent from somatostatin-containing neurons. Bimolecular-Fluorescence-Complementation identified D3 homodimers, a prerequisite for D3 activity, in processes of GT1-7 cells. Furthermore, T3-inducible D3 catalytic activity was detected in the rat median eminence. Triple-labeling immunofluorescence and immuno-electronmicroscopy revealed the presence of MCT8 on the surface of the vast majority of all types of hypophysiotropic terminals. The presence of MCT8 was also demonstrated on the axon terminals in the neurohaemal zone of the human infundibulum. The unexpected role of hypophysiotropic axons in fine-tuned regulation of T3 availability in these cells via MCT8-mediated transport and D3-catalyzed inactivation may represent a novel regulatory core mechanism for metabolism, growth, stress and reproduction in rodents and humans.

Deiodinases constitute a group of thioredoxin fold-containing selenoenzymes that play an important function in thyroid hormone homeostasis and control of thyroid hormone action. There are three known deiodinases: D1 and D2 activate the pro-hormone thyroxine (T4) to T3, the most active form of thyroid hormone, while D3 inactivates thyroid hormone and terminates T3 action. A number of studies indicate that deiodinase expression is altered in several types of cancers, suggesting that (i) they may represent a useful cancer marker and/or (ii) could play a role in modulating cell proliferation - in different settings thyroid hormone modulates cell proliferation. For example, although D2 is minimally expressed in human and rodent skeletal muscle, its expression level in rhabdomyosarcoma (RMS)-13 cells is threefold to fourfold higher. In basal cell carcinoma (BCC) cells, sonic hedgehog (Shh)-induced cell proliferation is accompanied by induction of D3 and inactivation of D2. Interestingly a fivefold reduction in the growth of BCC in nude mice was observed if D3 expression was knocked down. A decrease in D1 activity has been described in renal clear cell carcinoma, primary liver cancer, lung cancer, and some pituitary tumors, while in breast cancer cells and tissue there is an increase in D1 activity. Furthermore D1 mRNA and activity were found to be decreased in papillary thyroid cancer while D1 and D2 activities were significantly higher in follicular thyroid cancer tissue, in follicular adenoma, and in anaplastic thyroid cancer. It is conceivable that understanding how deiodinase dysregulation in tumor cells affect thyroid hormone signaling and possibly interfere with tumor progression could lead to new antineoplastic approaches.

Thyroid hormone action can be customized on a cell-specific fashion through the controlled action of the deiodinase group of enzymes, which are homodimeric thioredoxin fold containing selenoproteins. Whereas the type II deiodinase (D2) initiates thyroid hormone signaling by activating the pro-hormone thyroxine (T4) to the biologically active T3 molecule, the type III deiodinase (D3) terminates thyroid hormone action by catalyzing the inactivation of both T4 and T3 molecules. Deiodinases play a role in thyroid hormone homeostasis, development, growth and metabolic control by affecting the intracellular levels of T3 and thus gene expression on a cell-specific basis. Whereas both Dio2 and Dio3 are transcriptionally regulated, ubiquitination of D2 is a switch mechanism that controls D2 activity and intracellular T3 production. The hedgehog-inducible WSB-1 and the yeast Doa10 mammalian ortholog TEB4 are two E3 ligases that inactivate D2 via ubiquitination. Inactivation involves disruption of the D2:D2 dimer and can be reversed via two ubiquitin-specific proteases, USP20 and USP33, rescuing catalytic activity and T3 production. The ubiquitin-based switch mechanism that controls D2 activity illustrates how different cell types fine-tune thyroid hormone signaling, making D2 a suitable target for pharmacological intervention. This article reviews the cellular and molecular aspects of D2 regulation and the current models of D2-mediated thyroid hormone signaling.

The activities of malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G-6-PDH), two NADPH-generating lipogenic enzymes, were measured in brown adipose tissue (BAT) of rats undergoing various neurohormonal manipulations. Methimazole-induced hypothyroidism doubled the activity of these two enzymes but, surprisingly, triiodothyronine (T3) given to hypothyroid rats caused a time- and dose-dependent stimulation of up to three- to fourfold. Unilateral BAT denervation modestly reduced the activity of these enzymes (approximately 30%) and failed to prevent the stimulation induced by hypothyroidism, whereas growth hormone (GH) successfully blocked this effect of hypothyroidism. Insulin stimulated both enzymes regardless of the thyroid status but failed to abolish the inhibitory effect of GH. In intact rats, cold exposure caused a time-dependent increase in the activity of both ME and G-6-PDH, which reached 5.2- and 3-fold, respectively, after 96 h. This cold-induced stimulation was not observed in hypothyroid rats, but it was restored by physiological doses of thyroxine (800 ng.100 g body wt-1.24 h-1). Replacement with T3 (300 ng.100 g body wt-1.24 h-1), in contrast, did not have this effect. In hypothyroid rats with hemidenervation of BAT, norepinephrine (NE) modestly increased ME and G-6-PDH activities in the denervated side, with little or no effect in the intact side. Receptor-saturating doses of T3 (50 micrograms.100 g body wt-1.day-1 over 48 h) stimulated two- and threefold both enzymes in both sides, reducing or obliterating the effect of denervation. The data suggest a complex neurohormonal regulation of the activity of ME and G-6-PDH in BAT.(ABSTRACT TRUNCATED AT 250 WORDS)

It is widely accepted that thyroid hormone replacement for patients with hypothyroidism can be fully accomplished with levothyroxine monotherapy, as assessed by serum thyroid function tests. However, approximately 10% of hypothyroid patients are dissatisfied with the outcome of levothyroxine monotherapy, and physicians continue to report benefits from combined levothyroxine-triidothyronine therapy for some hypothyroid patients. Recently, a large prospective study reported that the benefit of the combined levothyroxine-triidothyronine therapy is associated with the Thr92Ala polymorphism in the type 2 deiodinase gene, which is present in about 15% of the general population. If confirmed, these findings indicate that personalized medicine is rapidly catching up with modern thyroidology.

The use of selective agonists of the thyroid hormone receptor isoform beta (TRbeta) has been linked to metabolic improvement in animal models of diet-induced obesity, nonalcoholic liver disease, and genetic hypercholesterolemia.To identify potential target tissues of such compounds, we exposed primary murine brown adipocytes and skeletal myocytes for 24 hours to 50 nM GC-24, a highly selective TRbeta agonist. GC-24 (17 ng/[g BW.day] for 36 days) was also tested in a mouse model of diet-induced obesity.While the brown adipocytes responded to GC-24, with 17%-400% increases in the expression of 12 metabolically relevant genes, the myocytes remained largely unresponsive to GC-24 treatment. In control mice kept on chow diet, GC-24 treatment accelerated energy expenditure by about 15% and limited body weight gain by about 50%. However, in the obese animals the GC-24-mediated reduction in body weight gain dropped to only 20%, while energy expenditure remained unaffected. In addition, an analysis of gene expression in the skeletal muscle, brown adipose tissue, and liver of these obese animals failed to identify a conclusive GC-24 transcriptome footprint.Feeding a high-fat diet impairs most of the beneficial metabolic effects associated with treatment with TRbeta-selective agonists.

We intend to develop in vitro model systems to study the hormonal regulation of uncoupling protein (UCP) and its role in brown adipose tissue (BAT) thermogenesis. We report here that UCP mRNA responses to adrenergic and thyroid hormone manipulations in freshly dispersed, mature brown adipocytes mimic in vivo observations. Studies were performed in brown adipocytes obtained from interscapular brown fat of euthyroid or hypothyroid rats. The tissue was dispersed with collagenase, and cells were isolated by floatation over 4% BSA. UCP mRNA in these cells is 2-3 times more abundant than that in the whole tissue, indicating a selection of cells expressing the gene. In cells from euthyroid rats, UCP mRNA is maximally elevated within 2 h of exposure to 1 microM forskolin and 50 ng T3/ml (77 nM total, 0.43 nM free). T3 significantly enhances the effect of forskolin. In the absence of stimulation, UCP mRNA rapidly disappears from euthyroid brown adipocytes, and this can be prevented with the addition of T3 by a mechanism not requiring on-going transcription. In cells from hypothyroid rats, forskolin or isoproterenol plus phenylephrine fail to stimulate UCP mRNA, but within 3 h of exposure to T3, cells recover full responsiveness. As in vivo, a high extracellular concentration of T3 is required for maximal responsiveness of UCP mRNA to cAMP, while T4 can restore responsiveness in physiological concentrations (40 pM). This effect of T4 is prevented by iopanoic acid, a compound that blocks the type II T4 5'-deiodinase. In conclusion, 1) freshly dispersed brown adipocytes retain all of the properties concerning UCP regulation by thyroid hormone and sympathetic nervous system described for brown fat in vivo; 2) the observations made in vivo, thus, represent direct action of norepinephrine and thyroid hormone on these cells; 3) as in vivo, T4 is a better source of intracellular T3 than extracellular T3 for brown adipocytes; hence, the in vivo findings are the result of the cell biology of 5'-deiodinase type II rather than dynamic factors inherent to the in vivo condition; 4) stabilization of mature UCP mRNA by T3 is an important mechanism to maintain the levels of this mRNA elevated under sustained stimulation; and 5) dispersed brown adipocytes and UCP gene products constitute a powerful model to study interactions between the sympathetic nervous system and thyroid hormone at a cellular or molecular level.

Hypothyroidism in humans is characterized by severe neurological consequences that are often irreversible, highlighting the critical role of thyroid hormone (TH) in the brain. Despite this, not much is known about the signaling pathways that control TH action in the brain. What is known is that the prohormone thyroxine (T4) is converted to the active hormone triiodothyronine (T3) by type 2 deiodinase (D2) and that this occurs in astrocytes, while TH receptors and type 3 deiodinase (D3), which inactivates T3, are found in adjacent neurons. Here, we modeled TH action in the brain using an in vitro coculture system of D2-expressing H4 human glioma cells and D3-expressing SK-N-AS human neuroblastoma cells. We found that glial cell D2 activity resulted in increased T3 production, which acted in a paracrine fashion to induce T3-responsive genes, including ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), in the cocultured neurons. D3 activity in the neurons modulated these effects. Furthermore, this paracrine pathway was regulated by signals such as hypoxia, hedgehog signaling, and LPS-induced inflammation, as evidenced both in the in vitro coculture system and in in vivo rat models of brain ischemia and mouse models of inflammation. This study therefore presents what we believe to be the first direct evidence for a paracrine loop linking glial D2 activity to TH receptors in neurons, thereby identifying deiodinases as potential control points for the regulation of TH signaling in the brain during health and disease.

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. In a setting of transient coexpression in HEK-293 cells, TEB4 and D2 could be coimmunoprecipitated, and additional TEB4 expression decreased D2 activity by approximately 50% (P0.05). A highly efficient TEB4 knockdown (90% reduction in mRNA and protein levels) decreased D2 ubiquitination and increased D2 activity and protein levels by about fourfold. The other activating deiodinase, D1, or a truncated D2 molecule (Delta18-D2) that lacks a critical instability domain was not affected by TEB4 knockdown. Furthermore, TEB4 knockdown prolonged D2 activity half-life at least fourfold, even under conditions known to promote D2 ubiquitination. Neither exposure to 1 microM of the proteasomal inhibitor MG132 for 24 h nor RNA interference WSB-1 knockdown resulted in additive effects on D2 expression when combined with TEB4 knockdown. Similar results were obtained with MSTO-211 cells, which endogenously express D2, after TEB4 knockdown using a lentivirus-based transduction strategy. While TEB4 expression predominates in the hematopoietic lineage, both WSB-1 and TEB4 are coexpressed with D2 in a number of tissues and cell types, except the thyroid and brown adipose tissue, where TEB4 expression is minimal. We conclude that TEB4 interacts with and mediates loss of D2 activity, indicating that D2 ubiquitination and degradation can be tissue specific, depending on WSB-1 and TEB4 expression levels.

Fatty liver is an important complication of obesity; however, regulatory mechanisms mediating altered gene expression patterns have not been identified.The aim of the study was to identify novel transcriptional changes in human liver that could contribute to hepatic lipid accumulation and associated insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis.We evaluated gene expression in surgical liver biopsies from 13 obese (nine with type 2 diabetes) and five control subjects using Affymetrix U133A microarrays. PCR validation was performed in liver biopsies using an additional 16 subjects. We also tested thyroid hormone responses in mice fed chow or high-fat diet.Recruitment was performed in an academic medical center.Individuals undergoing elective surgery for obesity or gallstones participated in the study.The top-ranking gene set, down-regulated in obese subjects, was comprised of genes previously demonstrated to be positively regulated by T(3) in human skeletal muscle (n = 399; P0.001; false discovery rate = 0.07). This gene set included genes related to RNA metabolism (SNRPE, HNRPH3, TIA1, and SFRS2), protein catabolism (PSMA1, PSMD12, USP9X, IBE2B, USP16, and PCMT1), and energy metabolism (ATP5C1, COX7C, UQCRB). We verified thyroid hormone regulation of these genes in the liver after injection of C57BL/6J mice with T(3) (100 microg/100 g body weight); furthermore, T(3)-induced increases in expression of these genes were abolished by high-fat diet. In agreement, expression of these genes inversely correlated with liver fat content in humans.These data suggest that impaired thyroid hormone action may contribute to altered patterns of gene expression in fatty liver.

Considerable indirect evidence suggests that the type 2 deiodinase (D2) generates T3 from T4 for local use in specific tissues including pituitary, brown fat, and brain, whereas the type I deiodinase (D1) generates T3 from T4 in the thyroid and peripheral tissues primarily for export to plasma. From studies in deiodinase-deficient mice, the importance of the D2 for local T3 generation has been confirmed. However, the phenotypes of these D1 knockout (KO) and D2KO mice are surprisingly mild and their serum T3 level, general health, and reproductive capacity are unimpaired. To explore further the importance of 5′deiodination in thyroid hormone economy, we used a mouse devoid of both D1 and D2 activity. In general, the phenotype of the D1/D2KO mouse is the sum of the phenotypes of the D1KO and D2KO mice. It appears healthy and breeds well, and most surprisingly its serum T3 level is normal. However, impairments in brain gene expression and possibly neurological function are somewhat greater than those seen in the D2KO mouse, and the serum rT3 level is elevated 6-fold in the D1/D2KO mouse but only 2-fold in the D1KO mouse and not at all in the D2KO mouse. The data suggest that whereas D1 and D2 are not essential for the maintenance of the serum T3 level, they do serve important roles in thyroid hormone homeostasis, the D2 being critical for local T3 production and the D1 playing an important role in iodide conservation by serving as a scavenger enzyme in peripheral tissues and the thyroid.

International audience; The multifactorial mechanisms promoting weight loss and improved metabolism following Roux-en-Y gastric bypass (GB) surgery remain incompletely understood. Recent rodent studies suggest that bile acids can mediate energy homeostasis by activating the G-protein coupled receptor TGR5 and the type 2 thyroid hormone deiodinase. Altered gastrointestinal anatomy following GB could affect enterohepatic recirculation of bile acids. We assessed whether circulating bile acid concentrations differ in patients who previously underwent GB, which might then contribute to improved metabolic homeostasis. We performed cross-sectional analysis of fasting serum bile acid composition and both fasting and post-meal metabolic variables, in three subject groups: (i) post-GB surgery (n = 9), (ii) without GB matched to preoperative BMI of the index cohort (n = 5), and (iii) without GB matched to current BMI of the index cohort (n = 10). Total serum bile acid concentrations were higher in GB (8.90 +/- 4.84 micromol/l) than in both overweight (3.59 +/- 1.95, P = 0.005, Ov) and severely obese (3.86 +/- 1.51, P = 0.045, MOb). Bile acid subfractions taurochenodeoxycholic, taurodeoxycholic, glycocholic, glycochenodeoxycholic, and glycodeoxycholic acids were all significantly higher in GB compared to Ov (P < 0.05). Total bile acids were inversely correlated with 2-h post-meal glucose (r = -0.59, P < 0.003) and fasting triglycerides (r = -0.40, P = 0.05), and positively correlated with adiponectin (r = -0.48, P < 0.02) and peak glucagon-like peptide-1 (GLP-1) (r = 0.58, P < 0.003). Total bile acids strongly correlated inversely with thyrotropic hormone (TSH) (r = -0.57, P = 0.004). Together, our data suggest that altered bile acid levels and composition may contribute to improved glucose and lipid metabolism in patients who have had GB.

Contains fulltext : 79580.pdf (Publisher’s version ) (Closed access) The cytosolic brain-type creatine kinase and mitochondrial ubiquitous creatine kinase (CK-B and UbCKmit) are expressed during the prepubescent and adult period of mammalian life. These creatine kinase (CK) isoforms are present in neural cell types throughout the central and peripheral nervous system and in smooth muscle containing tissues, where they have an important role in cellular energy homeostasis. Here, we report on the coupling of CK activity to body temperature rhythm and adaptive thermoregulation in mice. With both brain-type CK isoforms being absent, the body temperature reproducibly drops ~1.0 degrees C below normal during every morning (inactive) period in the daily cycle. Facultative non-shivering thermogenesis is also impaired, since CK--/-- mice develop severe hypothermia during 24 h cold exposure. A relationship with fat metabolism was suggested because comparison of CK--/-- mice with wildtype controls revealed decreased weight gain associated with less white and brown fat accumulation and smaller brown adipocytes. Also, circulating levels of glucose, triglycerides and leptin are reduced. Extensive physiological testing and uncoupling protein1 analysis showed, however, that the thermogenic problems are not due to abnormal responsiveness of brown adipocytes, since noradrenaline infusion produced a normal increase of body temperature. Moreover, we demonstrate that the cyclic drop in morning temperature is also not related to altered rhythmicity with reduced locomotion, diminished food intake or increased torpor sensitivity. Although several integral functions appear altered when CK is absent in the brain, combined findings point into the direction of inefficient neuronal transmission as the dominant factor in the thermoregulatory defect.

As judged by the response of uncoupling protein and key enzymes, brown adipose tissue (BAT) is highly dependent upon the local generation of T3 catalyzed by the tissue type II T4 5'-deiodinase (5'-D-II). In hypothyroid rats treated with T3 or T4, the capacity to withstand cold seems better correlated with the normalization of BAT responses than with the liver thyroid status. 5'D-II is activated by cold via sympathetic nervous system (SNS) stimulation, and the activation generates enough T3 to nearly saturate BAT nuclear T3 receptor (NTR) in euthyroid rats. In hypothyroidism, 5'D-II is highly stimulated by the SNS and hypothyroxinemia. In the present studies we have taken advantage of this situation to test 1) the capacity of 5'D-II to maintain nuclear T3 in rats with various degrees of hypothyroxinemia, and 2) the hypothesis that thyroid hormone-dependent BAT-facultative thermogenesis, rather than the effect of thyroid hormone on obligatory thermogenesis (basal metabolic rate), is the basic mechanism by which thyroid hormone confers protection against acute cold exposure. We treated methimazole-blocked rats (undetectable plasma T4 and T3) for a week with either subreplacement doses of T4 (0.5, 1, 2, and 4 micrograms/kg.day) or replacement doses of T4 or T3 (8 or 3 micrograms/kg.day, respectively). Sources and content of BAT nuclear T3 were studied at 25 C and after 48 h at 4 C by labeling the plasmaborne T3 (T3[T3]) with [131I]T3 and the locally generated T3 (T3[T4]) with [125I]T4. Neither the kinetics of nuclear-plasma exchange of T3[T3], the time of appearance of T3[T4] in BAT nuclei, nor NTR maximal binding capacity (0.71 ng T3/mg DNA) was affected by hypothyroidism. Kinetic analyses indicated a maximal BAT NTR occupancy of 40% at euthyroid serum T3 concentrations if T4 is not present. Replacement with T4 normalized both serum T4 and T3, while replacement with T3 normalized serum T3; for all other doses of T4, serum T4 and T3 concentrations were predictably related to the dose. 5'D-II activity decreased with increasing doses of T4, but for each dose of T4, this activity was 2-4 times greater at 4 C than at 25 C. BAT NTR occupancy normalized with 2 micrograms T4/kg in rats maintained at 25 C and with 4 micrograms T4/kg in cold-exposed rats, although in neither condition were serum T4 and T3 normalized nor more than 30% of NTR occupied by plasma T3.(ABSTRACT TRUNCATED AT 400 WORDS)

The iodothyronine deiodinases initiate or terminate thyroid hormone action and therefore are critical for the biological effects mediated by thyroid hormone. Over the years, research has focused on their role in preserving serum levels of the biologically active molecule T(3) during iodine deficiency. More recently, a fascinating new role of these enzymes has been unveiled. The activating deiodinase (D2) and the inactivating deiodinase (D3) can locally increase or decrease thyroid hormone signaling in a tissue- and temporal-specific fashion, independent of changes in thyroid hormone serum concentrations. This mechanism is particularly relevant because deiodinase expression can be modulated by a wide variety of endogenous signaling molecules such as sonic hedgehog, nuclear factor-kappaB, growth factors, bile acids, hypoxia-inducible factor-1alpha, as well as a growing number of xenobiotic substances. In light of these findings, it seems clear that deiodinases play a much broader role than once thought, with great ramifications for the control of thyroid hormone signaling during vertebrate development and metamorphosis, as well as injury response, tissue repair, hypothalamic function, and energy homeostasis in adults.

To explore the effect of refeeding on recovery of TRH gene expression in the hypothalamic paraventricular nucleus (PVN) and its correlation with the feeding-related neuropeptides in the arcuate nucleus (ARC), c-fos immunoreactivity (IR) in the PVN and ARC 2 h after refeeding and hypothalamic TRH, neuropeptide Y (NPY) and agouti-related protein (AGRP) mRNA levels 4, 12, and 24 h after refeeding were studied in Sprague-Dawley rats subjected to prolonged fasting. Despite rapid reactivation of proopiomelanocortin neurons by refeeding as demonstrated by c-fos IR in ARC α-MSH-IR neurons and ventral parvocellular subdivision PVN neurons, c-fos IR was present in only 9.7 ± 1.1% hypophysiotropic TRH neurons. Serum TSH levels remained suppressed 4 and 12 h after the start of refeeding, returning to fed levels after 24 h. Fasting reduced TRH mRNA compared with fed animals, and similar to TSH, remained suppressed at 4 and 12 h after refeeding, returning toward normal at 24 h. AGRP and NPY gene expression in the ARC were markedly elevated in fasting rats, AGRP mRNA returning to baseline levels 12 h after refeeding and NPY mRNA remaining persistently elevated even at 24 h. These data raise the possibility that refeeding-induced activation of melanocortin signaling exerts differential actions on its target neurons in the PVN, an early action directed at neurons that may be involved in satiety, and a later action on hypophysiotropic TRH neurons involved in energy expenditure, potentially mediated by sustained elevations in AGRP and NPY. This response may be an important homeostatic mechanism to allow replenishment of depleted energy stores associated with fasting.

The thyroid hormone plays a fundamental role in the development, growth, and metabolic homeostasis in all vertebrates by affecting the expression of different sets of genes. A group of thioredoxin fold-containing selenoproteins known as deiodinases control thyroid hormone action by activating or inactivating the precursor molecule thyroxine that is secreted by the thyroid gland. These pathways ensure regulation of the availability of the biologically active molecule T3, which occurs in a time-and tissue-specific fashion. In addition, because cells and plasma are in equilibrium and deiodination affects central thyroid hormone regulation, these local deiodinase-mediated events can also affect systemic thyroid hormone economy, such as in the case of non-thyroidal illness. Heightened interest in the field has been generated following the discovery that the deiodinases can be a component in both the Sonic hedgehog signaling pathway and the TGR-5 signaling cascade, a G-protein-coupled receptor for bile acids. These new mechanisms involved in deiodinase regulation indicate that local thyroid hormone activation and inactivation play a much broader role than previously thought.

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. Here we used fluorescence resonance energy transfer in live cells to demonstrate that D3 exists as homodimer. While D3 homodimerized in its native state, minor heterodimerization was also observed between D3:D1 and D3:D2 in intact cells, the significance of which remains elusive. Incubation with 0.5–1.2 m urea resulted in loss of D3 homodimerization as assessed by bioluminescence resonance energy transfer and a proportional loss of enzyme activity, to a maximum of approximately 50%. Protein modeling using a D2-based scaffold identified potential dimerization surfaces in the transmembrane and globular domains. Truncation of the transmembrane domain (ΔD3) abrogated dimerization and deiodinase activity except when coexpressed with full-length catalytically inactive deiodinase, thus assembled as ΔD3:D3 dimer; thus the D3 globular domain also exhibits dimerization surfaces. In conclusion, the inactivating deiodinase D3 exists as homo- or heterodimer in living intact cells, a feature that is critical for their catalytic activities.

The Sonic hedgehog (Shh) pathway plays a critical role in hair follicle physiology and is constitutively active in basal cell carcinomas (BCCs), the most common human malignancy. Type 3 iodothyronine deiodinase (D3), the thyroid hormone-inactivating enzyme, is frequently expressed in proliferating and neoplastic cells, but its role in this context is unknown. Here we show that Shh, through Gli2, directly induces D3 in proliferating keratinocytes and in mouse and human BCCs. We demonstrate that Gli-induced D3 reduces intracellular active thyroid hormone, thus resulting in increased cyclin D1 and keratinocyte proliferation. D3 knockdown caused a 5-fold reduction in the growth of BCC xenografts in nude mice. Shh-induced thyroid hormone degradation via D3 synergizes with the Shh-mediated reduction of the type 2 deiodinase, the thyroxine-activating enzyme, and both effects are reversed by cAMP. This previously unrecognized functional cross-talk between Shh/Gli2 and thyroid hormone in keratinocytes is a pathway by which Shh produces its proliferative effects and offers a potential therapeutic approach to BCC.

The type 2 deiodinase (D2) activates thyroid hormone and constitutes an important source of 3,5,3',-triiodothyronine in the brain. D2 is inactivated via WSB-1 mediated ubiquitination but can be rescued from proteasomal degradation by USP-33 mediated deubiquitination. Using an in silico analysis of published array data, we found a significant positive correlation between the relative mRNA expression levels of WSB-1 and USP-33 in a set of 56 mouse tissues (r = 0.08; P < 0.04). Subsequently, we used in situ hybridization combined with immunocytochemistry in rat brain to show that in addition to neurons, WSB-1 and USP-33 are differently expressed in astrocytes and tanycytes, the two main D2 expressing cell types in this tissue. Tanycytes, which are thought to participate in the feedback regulation of TRH neurons express both WSB-1 and USP-33, indicating the potential for D2 ubiquitination and deubiquitination in these cells. Notably, only WSB-1 is expressed in glial fibrillary acidic protein-positive astrocytes throughout the brain. Although developmental and environmental signals are known to regulate the expression of WSB-1 and USP-33 in other tissues, our real-time PCR studies indicate that changes in thyroid status do not affect the expression of these genes in several rat brain regions, whereas in the mediobasal hypothalamus, changes in gene expression were minimal. In conclusion, the correlation between the relative mRNA levels of WSB-1 and USP-33 in numerous tissues that do not express D2 suggests that these ubiquitin-related enzymes share additional substrates besides D2. Furthermore, the data indicate that changes in WSB-1 and USP-33 expression are not part of the brain homeostatic response to hypothyroidism or hyperthyroidism.

In humans, there is a significant decrease in serum T(3) and increase in rT(3) at different time points after myocardial infarction, whereas serum TSH and T(4) remain unaltered. We report here a time course study of pituitary-thyroid function and thyroid hormone metabolism in rats subjected to myocardial infarction by left coronary ligation (INF). INF- and sham-operated animals were followed by serial deiodination assays and thyroid function tests, just before, and 1, 4, 8, and 12 wk after surgery. At 4 and 12 wk after INF, liver type 1 deiodinase activity was significantly lower, confirming tissue hypothyroidism. Type 3 deiodinase (D3) activity was robustly induced 1 wk after INF only in the infarcted myocardium. Reminiscent of the consumptive hypothyroidism observed in patients with large D3-expressing tumors, this induction of cardiac D3 activity was associated with a decrease in both serum T(4) ( approximately 50% decrease) and T(3) (37% decrease), despite compensatory stimulation of the thyroid. Thyroid stimulation was documented by both hyperthyrotropinemia and radioiodine uptake. Serum TSH increased by 4.3-fold in the first and 3.1-fold in the fourth weeks (P < 0.01), returning to the basal levels thereafter. Thyroid sodium/iodide-symporter function increased 1 wk after INF, accompanying the increased serum TSH. We conclude that the acute decrease in serum T(4) and T(3) after INF is due to increased thyroid hormone catabolism from ectopic D3 expression in the heart.

The testis has been classically described as a thyroid hormone unresponsive tissue, but recent studies indicate that these hormones might play an important role in developing testes. We have previously demonstrated that type 2 iodothyronine deiodinase (D2), a thyroid hormone-activating enzyme, is expressed in adult rodent testis and that its activity is induced by hypothyroidism. Nevertheless, the precise location of D2 in testis is not known. The aim of the present work was to determine the testicular cell types in which D2 is expressed using real-time PCR analysis, in situ hybridization histochemistry, and determination of D2 activity in cell fractions isolated from adult euthyroid and/or hypothyroid rat testis. The D2 mRNA levels in germ cells were higher than those from somatic cells (6.94 ± 1.49 vs 2.32 ± 0.79 arbitrary units (au); P = 0.017). Hypothyroidism increased D2 expression in germ cells (6.94 ± 1.49 vs 8.78 ± 5.43 au, P = 0.002) but did not change D2 transcripts in somatic cells significantly (2.12 ± 0.79 vs 2.88 ± 1.39 au, P = 0.50). In situ hybridization analysis showed that D2 mRNA is specifically present in elongated spermatids undergoing differentiation, whereas other germ cell types and Sertoli cells of seminiferous epithelium and the interstitial cells were virtually negative for this enzyme. The enzyme activity measured in germ and somatic isolated cell fractions (0.23 ± 0.003 vs 0.02 ± 0.013 fmol/min per mg protein respectively; P < 0.001) further confirmed the real-time PCR and in situ hybridization results. Hence, our findings demonstrated that D2 is predominantly expressed in elongated spermatids, suggesting that thyroid hormone might have a direct effect on spermatogenesis in the adult rats.

Although platinum-based two-drug combinations represent the elective therapeutic approach for advanced/metastatic NSCLC, there is still interest in exploring the efficacy and tolerability of platinum-free combinations including third generation agents in selected NSCLC population. Based on the satisfying activity of gemcitabine (G), ifosfamide (I) and paclitaxel (T) as single agents in NSCLC, we have designed a phase II study to explore an alternative approach to platinum-containing regimens using a combination of these three drugs. To investigate the activity/toxicity of T 175 mg/m2 on day 1, I 3 g/m2 on day 1 (with Mesna uroprotection) and G 1,000 mg/m2 on day 1–8, every 3 weeks in the treatment of advanced/metastatic NSCLC, 46 patients (38 male, 8 female) with NSCLC were enrolled: mean age 58 (range 33–70); Stage IIIB/IV = 15/31; ECOG PS 0-1/2 = 31/15; Histology: adenocarcinoma = 20, squamous = 14, large cell = 3, NSCLC = 8, adenosquamous = 1. A total of 221 cycles have been administered (median number 4.8 for patients). In intent-to-treat analysis, partial response was achieved in 17 patients (36.95%), stable disease and progressive disease was detected in 16 (34.78%) and 10 (21.73%) patients, respectively. Time to progression was 30.9 weeks; median survival time was 42.7 weeks; the survival rates at 12 and 18 months were 34.79 and 15.21%, respectively. No toxic deaths occurred. No patients experienced grade 4 neutropenia and thrombocytopenia. Neutropenia grade 3 occurred in 10 patients (21.7%); Anemia grade 3 in 1 (2.1%); Thrombocytopenia grade 2 in two patients (4.3%) and grade 3 in one (2.1%). Peripheral neuropathy grade 1 occurred in ten (21.7%) and grade 2 in two patients (4.3%). Additional non-haematological toxicities were mild nausea, emesis and fatigue. GIT is well tolerated and active regimen in both advanced and metastatic NSCLC. These data suggest future investigations for GIT schedule as a possible alternative to platinum-based regimens in selected advanced/metastatic NSCLC patients where survival, tolerability and quality of life are the primary goals.

The three iodothyronine deiodinases catalyze the initiation (D1, D2) and termination (D3) of THyroid hormone effects in vertebrates. A 3-dimensional model predicts that these enzymes share a similar structural organization and belong to the thioredoxin (TRX) fold superfamily. Their active center is a selenocysteine-containing pocket defined by the β1-α1-β2 motifs of the TRX fold and a domain that shares strong similarities with the active site of iduronidase, a member of the clan GH-A fold of glycoside hydrolases. While D 1 and D3 are long-lived plasma membrane proteins (t 1/2 10–12 h), D2 is an endoplasmic reticulum resident protein that is inactivated by selective conjugation to ubiquitin, a process that is mediated by WSB-1, a Hedgehog-inducible gene. Remarkably, D2 ubiquitination is reversible and activity restored after deubiquitination by the pVHL-interacting deubiquitinating enzymes (VDU1 and VDU2). Deiodinases play a major role in development as well as in adults as critical players in Thyroid hormone homeostasis, particularly during hypo- and hyper Thyroidism. In addition to playing an important part in energy homeostasis, changes in deiodinase activity explain the alterations in Thyroid economy observed during illness and in the recently described syndrome of consumptive hypo Thyroidism.

We tested 413 water buffalo cows (142 cases and 271 controls) for the presence of anti-Brucella abortusantibodies (by the skin test, the agglutination test, and the complement fixation test) and theNramp1genotype (by capillary electrophoresis). Four alleles (Nramp1A, -B, -C, and -D) were detected in the 3′ untranslated region of theNramp1gene. TheBBgenotype was represented among only controls, providing evidence that this genotype confers resistance toBrucella abortus. The monocytes from the BB (resistant) subjects displayed a higher basal level ofNramp1mRNA and a lower number of viable intracellular bacteria than did the monocytes from AA (susceptible) subjects. The higher basal level of the antibacterial protein Nramp1 most probably provides the BB animals with the possibility of controlling bacteria immediately after their entry inside the cell.