Modulation of Deiodinase Types 2 and 3 during Skeletal Muscle Regeneration.

The muscle stem-cell niche comprises numerous cell types, which coordinate the regeneration process after injury. Thyroid hormones are one of the main factors that regulate genes linked to skeletal muscle. In this way, deiodinase types 2 and 3 are responsible for the fine-tuning regulation of the local T3 amount. Although their expression and activity have already been identified during muscle regeneration, it is of utmost importance to identify the cell type and temporal pattern of expression after injury to thoroughly comprehend their therapeutic potential. Here, we confirmed the expression of Dio2 and Dio3 in the whole tibialis anterior muscle. We identified, on a single-cell basis, that Dio2 is present in paired box 7 (PAX7)-positive cells starting from day 5 after injury. Dio2 is present in platelet derived growth factor subunit A (PDGFA)-expressing fibro-adipogenic progenitor cells between days 7 and 14 after injury. Dio3 is detected in myogenic differentiation (MYOD)-positive stem cells and in macrophages immediately post injury and thereafter. Interestingly, Dio2 and Dio3 RNA do not appear to be present in the same type of cell throughout the process. These results provide further insight into previously unseen aspects of the crosstalk and synchronized regulation of T3 in injured muscle mediated by deiodinases. The set of findings described here further define the role of deiodinases in muscle repair, shedding light on potential new forms of treatment for sarcopenia and other muscular diseases.

A systematic review of in vivo stretching regimens on inflammation and its relevance to translational yoga research.

OBJECTIVE: To conduct a systematic review evaluating the impact of stretching on inflammation and its resolution using in vivo rodent models. Findings are evaluated for their potential to inform the design of clinical yoga studies to assess the impact of yogic stretching on inflammation and health. METHODS: Studies were identified using four databases. Eligible publications included English original peer-reviewed articles between 1900-May 2020. Studies included those investigating the effect of different stretching techniques administered to a whole rodent model and evaluating at least one inflammatory outcome. Studies stretching the musculoskeletal and integumentary systems were considered. Two reviewers removed duplicates, screened abstracts, conducted full-text reviews, and assessed methodological quality. RESULTS: Of 766 studies identified, 25 were included for synthesis. Seven (28%) studies had a high risk of bias in 3 out of 10 criteria. Experimental stretching protocols resulted in a continuum of inflammatory responses with therapeutic and injurious effects, which varied with a combination of three stretching parameters--duration, frequency, and intensity. Relative to injurious stretching, therapeutic stretching featured longer-term stretching protocols. Evidence of pro- and mixed-inflammatory effects of stretching was found in 16 muscle studies. Evidence of pro-, anti-, and mixed-inflammatory effects was found in nine longer-term stretching studies of the integumentary system. CONCLUSION: Despite the overall high quality of these summarized studies, evaluation of stretching protocols paralleling yogic stretching is limited. Both injurious and therapeutic stretching induce aspects of inflammatory responses that varied among the different stretching protocols. Inflammatory markers, such as cytokines, are potential outcomes to consider in clinical yoga studies. Future translational research evaluating therapeutic benefits should consider in vitro studies, active vs. passive stretching, shorter-term vs. longer-term interventions, systemic vs. local effects of stretching, animal models resembling human anatomy, control and estimation of non-specific stresses, development of in vivo self-stretching paradigms targeting myofascial tissues, and in vivo models accounting for gross musculoskeletal posture.

Regulation of thyroid hormones and branchial iodothyronine deiodinases during freshwater acclimation in tilapia.

Euryhaline fishes are capable of maintaining osmotic homeostasis in a wide range of environmental salinities. Several pleiotropic hormones, including prolactin, growth hormone, and thyroid hormones (THs) are mediators of salinity acclimation. It is unclear, however, the extent to which THs and the pituitary-thyroid axis promote the adaptive responses of key osmoregulatory organs to freshwater (FW) environments. In the current study, we characterized circulating thyroxine (T4) and 3-3'-5-triiodothyronine (T3) levels in parallel with the outer ring deiodination (ORD) activities of deiodinases (dios) and mRNA expression of dio1, dio2, and dio3 in gill during the acclimation of Mozambique tilapia (Oreochromis mossambicus) to FW. Tilapia transferred from seawater (SW) to FW exhibited reduced plasma T4 and T3 levels at 6 h. These reductions coincided with an increase in branchial dio2-like activity and decreased branchial dio1 gene expression. To assess whether dios respond to osmotic conditions and/or systemic signals, gill filaments were exposed to osmolalities ranging from 280 to 450 mOsm/kg in an in vitro incubation system. Gene expression of branchial dio1, dio2, and dio3 was not directly affected by extracellular osmotic conditions. Lastly, we observed that dio1 and dio2 expression was stimulated by thyroid-stimulating hormone in hypophysectomized tilapia, suggesting that branchial TH metabolism is regulated by systemic signals. Our collective findings suggest that THs are involved in the FW acclimation of Mozambique tilapia through their interactions with branchial deiodinases that modulate their activities in a key osmoregulatory organ.

Thyroid Hormone Enhances Angiogenesis and the Warburg Effect in Squamous Cell Carcinomas.

Cancer angiogenesis is required to support energetic demand and metabolic stress, particularly during conditions of hypoxia. Coupled to neo-vasculogenesis, cancer cells rewire metabolic programs to sustain growth, survival and long-term maintenance. Thyroid hormone (TH) signaling regulates growth and differentiation in a variety of cell types and tissues, thus modulating hyper proliferative processes such as cancer. Herein, we report that TH coordinates a global program of metabolic reprogramming and induces angiogenesis through up-regulation of the VEGF-A gene, which results in the enhanced proliferation of tumor endothelial cells. In vivo conditional depletion of the TH activating enzyme in a mouse model of cutaneous squamous cell carcinoma (SCC) reduces the concentration of TH in the tumoral cells and results in impaired VEGF-A production and attenuated angiogenesis. In addition, we found that TH induces the expression of the glycolytic genes and fosters lactate production, which are key traits of the Warburg effect. Taken together, our results reveal a TH-VEGF-A-HIF1α regulatory axis leading to enhanced angiogenesis and glycolytic flux, which may represent a target for SCC therapy.

A Type 2 Deiodinase-Dependent Increase in Vegfa Mediates Myoblast-Endothelial Cell Crosstalk During Skeletal Muscle Regeneration.

Background: The type 2 deiodinase (DIO2) converts thyroxine to 3,3',5-triiodothyronine (T3), modulating intracellular T3. An increase in DIO2 within muscle stem cells during skeletal muscle regeneration leads to T3-dependent potentiation of differentiation. The muscle stem cell niche comprises numerous cell types, which coordinate the regeneration process. For example, muscle stem cells provide secretory signals stimulating endothelial cell-mediated vascular repair, and, in turn, endothelial cells promote muscle stem differentiation. We hypothesized that Dio2 loss in muscle stem cells directly impairs muscle stem cell-endothelial cell communication, leading to downstream disruption of endothelial cell function. Methods: We assessed the production of proangiogenic factors in differentiated C2C12 cells and in a C2C12 cell line without Dio2 (D2KO C2C12) by real-time quantitative-polymerase chain reaction and enzyme-linked immunosorbent assay. Conditioned medium (CM) was collected daily in parallel to evaluate its effects on human umbilical vein endothelial cell (HUVEC) proliferation, migration and chemotaxis, and vascular network formation. The effects of T3-treatment on vascular endothelial growth factor (Vegfa) mRNA expression in C2C12 cells and mouse muscle were assessed. Chromatin immunoprecipitation (ChIP) identified thyroid hormone receptor (TR) binding to the Vegfa gene. Using mice with a targeted disruption of Dio2 (D2KO mice), we determined endothelial cell number by immunohistochemistry/flow cytometry and evaluated related gene expression in both uninjured and injured skeletal muscle. Results: In differentiated D2KO C2C12 cells, Vegfa expression was 46% of wildtype (WT) C2C12 cells, while secreted VEGF was 45%. D2KO C2C12 CM exhibited significantly less proangiogenic effects on HUVECs. In vitro and in vivo T3 treatment of C2C12 cells and WT mice, and ChIP using antibodies against TRα, indicated that Vegfa is a direct genomic T3 target. In uninjured D2KO soleus muscle, Vegfa expression was decreased by 28% compared with WT mice, while endothelial cell numbers were decreased by 48%. Seven days after skeletal muscle injury, D2KO mice had 36% fewer endothelial cells, coinciding with an 83% decrease in Vegfa expression in fluorescence-activated cell sorting purified muscle stem cells. Conclusion:Dio2 loss in the muscle stem cell impairs muscle stem cell-endothelial cell crosstalk via changes in the T3-responsive gene Vegfa, leading to downstream impairment of endothelial cell function both in vitro and in vivo.

Establishment of a Novel Porcine Model to Study the Impact of Active Stretching on a Local Carrageenan-Induced Inflammation.

OBJECTIVE: Active stretching of the body is integral to complementary mind-body therapies such as yoga, as well as physical therapy, yet the biologic mechanisms underlying its therapeutic effects remain largely unknown. A previous study showed the impact of active stretching on inflammatory processes in rats. The present study tested the feasibility of using a porcine model, with a closer resemblance to human anatomy, to study the effects of active stretching in the resolution of localized inflammation. DESIGN: A total of 12 pigs were trained to stretch before subcutaneous bilateral Carrageenan injection in the back at the L3 vertebrae, 2 cm from the midline. Animals were randomized to no-stretch or stretch, twice a day for 5 mins over 48 hrs. Animals were euthanized for tissue collection 48 hrs postinjection. RESULTS: The procedure was well tolerated by the pigs. On average, lesion area was significantly smaller by 36% in the stretch group compared with the no-stretch group (P = 0.03). CONCLUSION: This porcine model shows promise for studying the impact of active stretching on inflammation-resolution mechanisms. These results are relevant to understanding the stretching-related therapeutic mechanisms of mind-body therapies. Future studies with larger samples are warranted.

The thyroid hormone activating enzyme, type 2 deiodinase, induces myogenic differentiation by regulating mitochondrial metabolism and reducing oxidative stress.

Thyroid hormone (TH) is a key metabolic regulator that acts by coordinating short- and long-term energy needs. Accordingly, significant metabolic changes are observed depending on thyroid status. Although it is established that hyperthyroidism augments basal energy consumption, thus resulting in an enhanced metabolic state, the net effects on cellular respiration and generation of reactive oxygen species (ROS) remain unclear. To elucidate the effects of augmented TH signal in muscle cells, we generated a doxycycline-inducible cell line in which the expression of the TH-activating enzyme, type 2 deiodinase (D2), is reversibly turned on by the "Tet-ON" system. Interestingly, increased intracellular TH caused a net shift from oxidative phosphorylation to glycolysis and a consequent increase in the extracellular acidification rate. As a result, mitochondrial ROS production, and both the basal and doxorubicin-induced production of cellular ROS were reduced. Importantly, the expression of a set of antioxidant genes was up-regulated, and, among them, the mitochondrial scavenger Sod2 was specifically induced at transcriptional level by D2-mediated TH activation. Finally, we observed that attenuation of oxidative stress and increased levels of SOD2 are key elements of the differentiating cascade triggered by TH and D2, thereby establishing that D2 is essential in coordinating metabolic reprogramming of myocytes during myogenic differentiation. In conclusion, our findings indicate that TH plays a key role in oxidative stress dynamics by regulating ROS generation. Our novel finding that TH and its intracellular metabolism act as mitochondrial detoxifying agents sheds new light on metabolic processes relevant to muscle physiology.

A Global Loss of Dio2 Leads to Unexpected Changes in Function and Fiber Types of Slow Skeletal Muscle in Male Mice.

The type 2 iodothyronine-deiodinase (D2) enzyme converts T4 to T3, and mice deficient in this enzyme [D2 knockout (D2KO) mice] have decreased T3 derived from T4 in skeletal muscle despite normal circulating T3 levels. Because slow skeletal muscle is particularly susceptible to changes in T3 levels, we expected D2 inactivation to result in more pronounced slow-muscle characteristics in the soleus muscle, mirroring hypothyroidism. However, ex vivo studies of D2KO soleus revealed higher rates of twitch contraction and relaxation and reduced resistance to fatigue. Immunostaining of D2KO soleus showed that these properties were associated with changes in muscle fiber type composition, including a marked increase in the number of fast, glycolytic type IIB fibers. D2KO soleus muscle fibers had a larger cross-sectional area, and this correlated with increased myonuclear accretion in myotubes formed from D2KO skeletal muscle precursor cells differentiated in vitro. Consistent with our functional findings, D2KO soleus gene expression was markedly different from that in hypothyroid wild-type (WT) mice. Comparison of gene expression between euthyroid WT and D2KO mice indicated that PGC-1α, a T3-dependent regulator of slow muscle fiber type, was decreased by ∼50% in D2KO soleus. Disruption of Dio2 in the C2C12 myoblast cell line led to a significant decrease in PGC-1α expression and a faster muscle phenotype upon differentiation. These results indicate that D2 loss leads to significant changes in soleus contractile function and fiber type composition that are inconsistent with local hypothyroidism and suggest that reduced levels of PCG-1α may contribute to the observed phenotypical changes.

Quantitative Analysis of the Benefits and Risk of Thyroid Nodule Evaluation in Patients ≥70 Years Old.

BACKGROUND: In older patients, thyroid nodules are frequently detected and referred for evaluation, though usually prove to be benign disease or low-risk cancer. Therefore, management should be guided not solely by malignancy risk, but also by the relative risks of any intervention. Unfortunately, few such data are available for patients ≥70 years old. METHODS: All consecutive patients ≥70 years old assessed by ultrasound (US) and fine-needle aspiration (FNA) between 1995 and 2015 were analyzed. Clinical, US, and histologic data, including patient comorbidities and outcomes, were obtained. Imaging and cytology results from initial evaluation were reviewed to detect significant-risk thyroid cancer (SRTC), which was defined as anaplastic, medullary, or poorly differentiated carcinoma, or the presence of distant metastases. Overall survival analyses were then performed to assist with risk-to-benefit assessment. RESULTS: A total of 1129 patients ≥70 years old with 2527 nodules ≥1 cm were evaluated. FNA was safe in all, and cytology proved benign in 67.3% of patients. However, FNA led to surgery in 208 patients, of whom 93 (44.7%) had benign histopathology. Among all patients who underwent FNA, only 17 (1.5%) SRTC were identified, all of which were preoperatively identifiable by imaging and/or cytology. These SRTC were responsible for all (n = 10; 0.9%) thyroid cancer deaths. Among all other patients (n = 1112), 160 deaths (14.4%) were confirmed during a median follow-up of four years. None of these were thyroid cancer related. Survival analysis for these 1112 patients demonstrated that a separate non-thyroidal malignancy or coronary artery disease at the time of nodule evaluation was associated with increased mortality compared to those without these diagnoses (hazard ratio = 2.32 [confidence interval 1.66-3.26]; p < 0.01), confirming these are important variables to identify prior to thyroid nodule evaluation. CONCLUSIONS: For patients ≥70 years old, US and FNA are safe and prove helpful in identifying SRTC and benign cytology. However, the surgical management of patients ≥70 years old presenting without high-risk findings should be tempered, especially when comorbid illness is identified.

T3 Induces Both Markers of Maturation and Aging in Pancreatic ß-Cells.

Previously, we showed that thyroid hormone (TH) triiodothyronine (T3) enhanced ß-cell functional maturation through induction of Mafa High levels of T3 have been linked to decreased life span in mammals and low levels to lengthened life span, suggesting a relationship between TH and aging. Here, we show that T3 increased p16Ink4a (a ß-cell senescence marker and effector) mRNA in rodent and human ß-cells. The kinetics of Mafa and p16Ink4a induction suggested both genes as targets of TH via TH receptors (THRs) binding to specific response elements. Using specific agonists CO23 and GC1, we showed that p16Ink4a expression was controlled by THRA and Mafa by THRB. Using chromatin immunoprecipitation and a transient transfection yielding biotinylated THRB1 or THRA isoforms to achieve specificity, we determined that THRA isoform bound to p16Ink4a , whereas THRB1 bound to Mafa but not to p16Ink4a On a cellular level, T3 treatment accelerated cell senescence as shown by increased number of ß-cells with acidic ß-galactosidase activity. Our data show that T3 can simultaneously induce both maturation (Mafa) and aging (p16Ink4a ) effectors and that these dichotomous effects are mediated through different THR isoforms. These findings may be important for further improving stem cell differentiation protocols to produce functional ß-cells for replacement therapies in diabetes.

Differential Growth Rates of Benign vs. Malignant Thyroid Nodules.

Context: Thyroid nodule growth was once considered concerning for malignancy, but data showing that benign nodules grow questioned the use of this paradigm. To date, however, no studies have adequately evaluated whether growth rates differ in malignant vs. benign nodules. Objective: To sonographically evaluate growth rates in benign and malignant thyroid nodules ≥1 cm. Design: Prospective, cohort study of patients with tissue diagnosis of benign or malignant disease, with repeated ultrasound evaluation six or more months apart. Main Outcomes: Growth rate in largest dimension of malignant compared with benign thyroid nodules. Regression models were used to evaluate predictors of growth. Results: Malignant nodules (126) met inclusion criteria (≥6-month nonoperative followup) and were compared with 1363 benign nodules. Malignant nodules were not found to be uniquely selected or prospectively observed solely for low-risk phenotype. Median ultrasound intervals were similar (21.8 months for benign nodules; 20.9 months for malignant nodules). Malignant nodules were more likely to grow >2 mm/y compared with benign nodules [relative risk (RR) = 2.5, 95% confidence interval (CI), 1.6 to 3.1; P < 0.001], which remained true after adjustment for clinical factors. The RR of a nodule being malignant increased with faster growth rates. Malignant nodules growing >2 mm/y had greater odds of being more aggressive cancers [intermediate risk: odds ratio (OR) = 2.99; 95% CI, 1.20 to 7.47; P = 0.03; higher risk: OR = 8.69; 95% CI, 1.78 to 42.34; P = 0.02]. Conclusions: Malignant nodules, especially higher-risk phenotypes, grow faster than benign nodules. As growth >2 mm/y predicts malignant compared with benign disease, this clinical parameter can contribute to the assessment of thyroid cancer risk.

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. There was no growth or reproductive phenotype. Basal TSH levels were increased 1.5- to 1.8-fold, but serum T4 and T3 were not different from the controls in adult mice. In hypothyroid adult mice, suppression of TSH by T4, but not T3, was impaired. Despite mild basal TSH elevation, the TSH increase in response to hypothyroidism was 4-fold reduced in the Cga-cre D2KO compared with control mice despite an identical level of pituitary TSH α- and ß-subunit mRNAs. In neonatal Cga-cre D2KO mice, TSH was also 2-fold higher than in the controls, but serum T4 was elevated. Despite a constant TSH, serum T4 increased 2-3-fold between postnatal day (P) 5 and P15 in both genotypes. The pituitary, but not cerebrocortical, D2 activity was markedly elevated in P5 mice decreasing towards adult levels by P17. In conclusion, a congenital severe reduction of thyrotroph D2 causes a major impairment of the TSH response to hypothyroidism. This would be deleterious to the compensatory adaptation of the thyroid gland to iodine deficiency.

Repair-related activation of hedgehog signaling in stromal cells promotes intrahepatic hypothyroidism.

Thyroid hormone (TH) is important for tissue repair because it regulates cellular differentiation. Intrahepatic TH activity is controlled by both serum TH levels and hepatic deiodinases. TH substrate (T4) is converted into active hormone (T3) by deiodinase 1 (D1) but into inactive hormone (rT3) by deiodinase 3 (D3). Although the relative expressions of D1 and D3 are known to change during liver injury, the cell types and signaling mechanisms involved are unclear. We evaluated the hypothesis that changes in hepatic deiodinases result from repair-related activation of the Hedgehog pathway in stromal cells. We localized deiodinase expression, assessed changes during injury, and determined how targeted manipulation of Hedgehog signaling in stromal cells impacted hepatic deiodinase expression, TH content, and TH action in rodents. Humans with chronic liver disease were also studied. In healthy liver, hepatocytes strongly expressed D1 and stromal cells weakly expressed D3. During injury, hepatocyte expression of D1 decreased, whereas stromal expression of D3 increased, particularly in myofibroblasts. Conditionally disrupting Hedgehog signaling in myofibroblasts normalized deiodinase expression. Repair-related changes in deiodinases were accompanied by reduced hepatic TH content and TH-regulated gene expression. In patients, this was reflected by increased serum rT3. Moreover, the decreases in the free T3 to rT3 and free T4 to rT3 ratios distinguished advanced from mild fibrosis, even in individuals with similar serum levels of TSH and free T4. In conclusion, the Hedgehog-dependent changes in liver stromal cells drive repair-related changes in hepatic deiodinase expression that promote intrahepatic hypothyroidism, thereby limiting exposure to T3, an important factor for cellular differentiation.

Thyroid hormones and skeletal muscle--new insights and potential implications.

Thyroid hormone signalling regulates crucial biological functions, including energy expenditure, thermogenesis, development and growth. The skeletal muscle is a major target of thyroid hormone signalling. The type 2 and 3 iodothyronine deiodinases (DIO2 and DIO3, respectively) have been identified in skeletal muscle. DIO2 expression is tightly regulated and catalyses outer-ring monodeiodination of the secreted prohormone tetraiodothyronine (T4) to generate the active hormone tri-iodothyronine (T3). T3 can remain in the myocyte to signal through nuclear receptors or exit the cell to mix with the extracellular pool. By contrast, DIO3 inactivates T3 through removal of an inner-ring iodine. Regulation of the expression and activity of deiodinases constitutes a cell-autonomous, pre-receptor mechanism for controlling the intracellular concentration of T3. This local control of T3 activity is crucial during the various phases of myogenesis. Here, we review the roles of T3 in skeletal muscle development and homeostasis, with a focus on the emerging local deiodinase-mediated control of T3 signalling. Moreover, we discuss these novel findings in the context of both muscle homeostasis and pathology, and examine how skeletal muscle deiodinase activity might be therapeutically harnessed to improve satellite-cell-mediated muscle repair in patients with skeletal muscle disorders, muscle atrophy or injury.

Thyroid hormone promotes postnatal rat pancreatic ß-cell development and glucose-responsive insulin secretion through MAFA.

Neonatal ß cells do not secrete glucose-responsive insulin and are considered immature. We previously showed the transcription factor MAFA is key for the functional maturation of ß cells, but the physiological regulators of this process are unknown. Here we show that postnatal rat ß cells express thyroid hormone (TH) receptor isoforms and deiodinases in an age-dependent pattern as glucose responsiveness develops. In vivo neonatal triiodothyronine supplementation and TH inhibition, respectively, accelerated and delayed metabolic development. In vitro exposure of immature islets to triiodothyronine enhanced the expression of Mafa, the secretion of glucose-responsive insulin, and the proportion of responsive cells, all of which are effects that were abolished in the presence of dominant-negative Mafa. Using chromatin immunoprecipitation and electrophoretic mobility shift assay, we show that TH has a direct receptor-ligand interaction with the Mafa promoter and, using a luciferase reporter, that this interaction was functional. Thus, TH can be considered a physiological regulator of functional maturation of ß cells via its induction of Mafa.

A novel pathway regulates thyroid hormone availability in rat and human hypothalamic neurosecretory neurons.

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.

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. Endogenous D1 activity in sonicates was inactivated in a dose-dependent manner in HepG2 cells, with a ∼50% decrease after 10 nM rT(3) treatment. Inactivation of D1 was rapid, occurring after only half an hour of rT(3) treatment. D1 expressed in HEK293 cells was inactivated by rT(3) in a similar manner. (75)Se labeling of the D1 selenoprotein indicated that after 4 h rT(3)-mediated inactivation of D1 occurs without a corresponding decrease in D1 protein levels, though rT(3) treatment causes a loss of D1 protein after 8-24 h. Bioluminescence resonance energy transfer studies indicate that rT(3) exposure increases energy transfer between the D1 homodimer subunits, and this was lost when the active site of D1 was mutated to alanine, suggesting that a post-catalytic structural change in the D1 homodimer could cause enzyme inactivation. Thus, both D1 and type 2 deiodinase are subject to catalysis-induced loss of activity although their inactivation occurs via very different mechanisms.

The role of the iodothyronine deiodinases in the physiology and pathophysiology of thyroid hormone action.

Thyroxine (T4) is a prohormone and must be activated to 3,5,3' triodothyronine (T3) by either the type 1 (D1) or 2 (D2) selenodeiodinase. A third deiodinase (D3) inactivates T3 or T4 by removal of an inner ring iodine. These reactions require both a deiodinase enzyme and a cofactor, probably a thiol, to reduce the oxidized selenolyl group in the active center of each deiodinase. Thus, deiodination rates depend on both the enzyme and cofactor. The source of most of the circulating T3 is D1-mediated, while D2 provides nuclear receptor-bound hormone. Using sensitive and specific assays, it has become apparent that both D2 and D3 are widespread throughout vertebrate tissues. The complex interactions between the activating D2 and the inactivating D3 in tissues expressing these two enzymes determine the intracellular T3 concentration. This provides enormous flexibility for both developmental and tissue regeneration processes, allowing exquisite control of intracellular T3 concentrations. The endogenous factors regulating the activity of these enzymes, such as the hedgehog proteins, FoxO3, or the wnt/ß catenin pathway together with the actions of thyroid hormone transporters, direct adjustments of nuclear receptor bound T3 which in turn can control the balance between cellular proliferation and differentiation. Their actions provide dynamic flexibility to what appears on the surface to be a very static hormonal system.

The arrestin domain-containing 3 protein regulates body mass and energy expenditure.

A human genome-wide linkage scan for obesity identified a linkage peak on chromosome 5q13-15. Positional cloning revealed an association of a rare haplotype to high body-mass index (BMI) in males but not females. The risk locus contains a single gene, "arrestin domain-containing 3" (ARRDC3), an uncharacterized α-arrestin. Inactivating Arrdc3 in mice led to a striking resistance to obesity, with greater impact on male mice. Mice with decreased ARRDC3 levels were protected from obesity due to increased energy expenditure through increased activity levels and increased thermogenesis of both brown and white adipose tissues. ARRDC3 interacted directly with ß-adrenergic receptors, and loss of ARRDC3 increased the response to ß-adrenergic stimulation in isolated adipose tissue. These results demonstrate that ARRDC3 is a gender-sensitive regulator of obesity and energy expenditure and reveal a surprising diversity for arrestin family protein functions.