Uncovering Actions of Type 3 Deiodinase in the Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD).

Metabolic dysfunction-associated fatty liver disease (MAFLD) has gained worldwide attention as a public health problem. Nonetheless, lack of enough mechanistic knowledge restrains effective treatments. It is known that thyroid hormone triiodothyronine (T3) regulates hepatic lipid metabolism, and mitochondrial function. Liver dysfunction of type 3 deiodinase (D3) contributes to MAFLD, but its role is not fully understood. OBJECTIVE: To evaluate the role of D3 in the progression of MAFLD in an animal model. METHODOLOGY: Male/adult Sprague Dawley rats (n = 20) were allocated to a control group (2.93 kcal/g) and high-fat diet group (4.3 kcal/g). Euthanasia took place on the 28th week. D3 activity and expression, Uncoupling Protein 2 (UCP2) and type 1 deiodinase (D1) expression, oxidative stress status, mitochondrial, Krebs cycle and endoplasmic reticulum homeostasis in liver tissue were measured. RESULTS: We observed an increase in D3 activity/expression (p < 0.001) related to increased thiobarbituric acid reactive substances (TBARS) and carbonyls and diminished reduced glutathione (GSH) in the MAFLD group (p < 0.05). There was a D3-dependent decrease in UCP2 expression (p = 0.01), mitochondrial capacity, respiratory activity with increased endoplasmic reticulum stress in the MAFLD group (p < 0.001). Surprisingly, in an environment with lower T3 levels due to high D3 activity, we observed an augmented alpha-ketoglutarate dehydrogenase (KGDH) and glutamate dehydrogenase (GDH) enzymes activity (p < 0.05). CONCLUSION: Induced D3, triggered by changes in the REDOX state, decreases T3 availability and hepatic mitochondrial capacity. The Krebs cycle enzymes were altered as well as endoplasmic reticulum stress. Taken together, these results shed new light on the role of D3 metabolism in MAFLD.

Thyroid hormones: Metabolism and transportation in the fetoplacental unit.

The thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3), are of vital importance for fetal development. The concentration of THs in fetal circulation varies throughout gestation and differs from the concentration in the maternal serum, indicating the presence of maternal-fetal thyroid homeostasis regulatory mechanisms in the placenta. The passage of THs from maternal circulation to fetal circulation is modulated by plasma membrane transporters, enzymes, and carrier proteins. Monocarboxylate transporter 8, iodothyronine deiodinases (DIO2 and DIO3), and transthyretin are especially involved in this maternal-fetal thyroid modulation, shown by a greater expression in the placenta. THs also play a role in placental development and as expected, abnormal variations in TH levels are associated with pregnancy complications and can result in damage to the fetus. Although new evidence regarding TH regulation during pregnancy and its effects in the mother, placenta, and fetus has been published, many aspects of these interactions are still poorly understood. The objective of this review is to provide an evidence-based update, drawn from current data, on the metabolism and transport of THs in the placenta and their vital role in the maternal-fetal relationship.

Liraglutide Activates Type 2 Deiodinase and Enhances ß3-Adrenergic-Induced Thermogenesis in Mouse Adipose Tissue.

Aims: Liraglutide is a long-acting glucagon-like peptide 1 (GLP-1) receptor agonist used as an anti-hyperglycemic agent in type 2 diabetes treatment and recently approved for obesity management. Weight loss is attributed to appetite suppression, but therapy may also increase energy expenditure. To further investigate the effect of GLP-1 signaling in thermogenic fat, we assessed adipose tissue oxygen consumption and type 2 deiodinase (D2) activity in mice treated with liraglutide, both basally and after ß3-adrenergic treatment. Methods: Male C57BL/6J mice were randomly assigned to receive liraglutide (400 µg/kg, n=12) or vehicle (n=12). After 16 days, mice in each group were co-treated with the selective ß3-adrenergic agonist CL316,243 (1 mg/kg, n=6) or vehicle (n=6) for 5 days. Adipose tissue depots were assessed for gene and protein expression, oxygen consumption, and D2 activity. Results: Liraglutide increased interscapular brown adipose tissue (iBAT) oxygen consumption and enhanced ß3-adrenergic-induced oxygen consumption in iBAT and inguinal white adipose tissue (ingWAT). These effects were accompanied by upregulation of UCP-1 protein levels in iBAT and ingWAT. Notably, liraglutide increased D2 activity without significantly upregulating its mRNA levels in iBAT and exhibited additive effects to ß3-adrenergic stimulation in inducing D2 activity in ingWAT. Conclusions: Liraglutide exhibits additive effects to those of ß3-adrenergic stimulation in thermogenic fat and increases D2 activity in BAT, implying that it may activate this adipose tissue depot by increasing intracellular thyroid activation, adding to the currently known mechanisms of GLP-1A-induced weight loss.

Thyroxine replacement modifies changes in deiodinase and thyroid hormone transporter expression induced by subclinical hypothyroidism in rats.

PURPOSE: The potential benefits of treating subclinical hypothyroidism (SCH) are unclear and still controversial. Thus, we surgically induced SCH in rats and evaluated the effects of thyroxine (T4) replacement on the gene expression levels of deiodinases and thyroid hormone (TH) transporters in different tissues. METHODS: SCH was induced by hemithyroid electrocauterization. The control animals underwent the same surgical procedure but were not subjected to electrocauterization (sham). After 14 days, half of the SCH animals were treated with T4 (SCH + T4). At the end of the experimental protocol, all of the rats were euthanized, serum hormone concentrations were measured, and RNA analyses were performed on different tissues and organs. RESULTS: Consistent with previous studies, we observed increased TSH levels, normal TH levels, and reduced hypothalamic TRH expression in the SCH group. Additionally, Dio2 mRNA expression was downregulated in the hippocampus and pituitary, and Dio1 was upregulated in the kidney and pituitary of the SCH animals. The changes in Dio3 expression were tissue-specific. Concerning TH transporters, Mct10 expression was upregulated in the pituitary, kidney, hypothalamus, and hippocampus, and Mct8 expression was downregulated in the kidney of the SCH group. Crym expression was upregulated in the kidney and pituitary. Notably, T4 replacement significantly attenuated serum TSH levels and reverted Dio1, Dio2, Mct10, and Crym expression in the pituitary, hippocampus, and kidney to levels that were similar to the sham group. Tissue-specific responses were also observed in the liver and hypothalamus. CONCLUSION: Our results indicate that treatment of SCH should be considered before the appearance of clinical symptoms of hypothyroidism.

Decreased expression of the thyroid hormone-inactivating enzyme type 3 deiodinase is associated with lower survival rates in breast cancer.

Thyroid hormones (THs) are critical regulators of cellular processes, while changes in their levels impact all the hallmarks of cancer. Disturbed expression of type 3 deiodinase (DIO3), the main TH-inactivating enzyme, occurs in several human neoplasms and has been associated with adverse outcomes. Here, we investigated the patterns of DIO3 expression and its prognostic significance in breast cancer. DIO3 expression was evaluated by immunohistochemistry in a primary cohort of patients with breast cancer and validated in a second cohort using RNA sequencing data from the TCGA database. DNA methylation data were obtained from the same database. DIO3 expression was present in normal and tumoral breast tissue. Low levels of DIO3 expression were associated with increased mortality in the primary cohort. Accordingly, low DIO3 mRNA levels were associated with an increased risk of death in a multivariate model in the validation cohort. DNA methylation analysis revealed that the DIO3 gene promoter is hypermethylated in tumors when compared to normal tissue. In conclusion, DIO3 is expressed in normal and tumoral breast tissue, while decreased expression relates to poor overall survival in breast cancer patients. Finally, loss of DIO3 expression is associated with hypermethylation of the gene promoter and might have therapeutic implications.

Sepsis Impairs Thyroid Hormone Signaling and Mitochondrial Function in the Mouse Diaphragm.

Background: Sepsis can cause the nonthyroidal illness syndrome (NTIS), resulting in perturbed thyroid hormone (TH) signaling and reduced thyroxine (T4) levels. TH is a major regulator of muscle function, via its influence on mitochondria. This study aimed at evaluating the relationship between TH signaling, mitochondrial function, and the antioxidant defense system in the diaphragms of septic mice. Methods: Male C57Bl/6 mice were divided into two groups: cecal ligation and puncture (CLP) and sham. Twenty-four hours after surgery, plasma, diaphragms, and livers were collected. TH metabolism and responses were analyzed by measuring messenger RNA (mRNA) expression of Dio1 in the liver, and Thra, Thrb, Dio2, Slc16a10, and Slc16a2 (encodes MCT 10 and 8), in the diaphragm. T4 plasma levels were measured by radioimmunoassay. Damage to diaphragm mitochondria was assessed by electron microscopy and real-time polymerase chain reaction (qPCR), and function with oxygraphy. The diaphragm antioxidative defense system was examined by qPCR, analyzing superoxide dismutase (SOD) 1 (Sod1), mitochondrial superoxide dismutase (SOD 2; Sod2), extracellular superoxide dismutase (SOD 3; Sod3), glutathione peroxidase 1 (Gpx1), and catalase (Cat) expression. The effect of TH replacement was tested by treating the mice with T4 and triiodothyronine (T3) (CLP+TH) after surgery. Results: CLP mice presented reduced total plasma T4 concentrations, downregulated Dio1, and upregulated Il1b mRNA expression in the liver. CLP mice also displayed downregulated Thra, Thrb, Slc16a10, and Slc16a2 expression in the diaphragm, suggesting that TH signaling was compromised. The expression of Ppargc1a (encoding PGC1a) was downregulated, which correlated with the decrease in the number of total mitochondria, increase in the percentage of injured mitochondria, downregulation of respiratory chain complex 2 and 3 mRNA expression, and reduced maximal respiration. In addition, septic animals presented a three-fold increase in Ucp3 and G6pdh expression; downregulated Sod3, Gpx1, and Cat expression; and upregulated Sod2 expression, potentially due to elevated reactive oxygen species levels. The mitochondrial number and the percentage of injured mitochondrial were similar between sham and CLP+TH mice. Conclusions: Sepsis induced responses consistent with NTIS, resulted in mitochondrial damage and functional impairment, and modulated the expression of key antioxidant enzymes in the diaphragm. Thus, impaired diaphragm function during sepsis seems to involve altered local TH signaling, mitochondrial dysfunction, and oxidative stress defense.

Propranolol inhibits myocardial infarction-induced brown adipose tissue D2 activation and maintains a low thyroid hormone state in rats.

Considering the recognized role of thyroid hormones on the cardiovascular system during health and disease, we hypothesized that type 2 deiodinase (D2) activity, the main activation pathway of thyroxine (T4)-to-triiodothyronine (T3), could be an important site to modulate thyroid hormone status, which would then constitute a possible target for ß-adrenergic blocking agents in a myocardial infarction (MI) model induced by left coronary occlusion in rats. Despite a sustained and dramatic fall in serum T4 concentrations (60-70%), the serum T3 concentration fell only transiently in the first week post-infarction (53%) and returned to control levels at 8 and 12 weeks after surgery compared to the Sham group (P<0.05). Brown adipose tissue (BAT) D2 activity (fmol T4·min-1·mg ptn-1) was significantly increased by approximately 77% in the 8th week and approximately 100% in the 12th week in the MI group compared to that of the Sham group (P<0.05). Beta-blocker treatment (0.5 g/L propranolol given in the drinking water) maintained a low T3 state in MI animals, dampening both BAT D2 activity (44% reduction) and serum T3 (66% reduction in serum T3) compared to that of the non-treated MI group 12 weeks after surgery (P<0.05). Propranolol improved cardiac function (assessed by echocardiogram) in the MI group compared to the non-treated MI group by 40 and 57%, 1 and 12 weeks after treatment, respectively (P<0.05). Our data suggested that the beta-adrenergic pathway may contribute to BAT D2 hyperactivity and T3 normalization after MI in rats. Propranolol treatment maintained low T3 state and improved cardiac function additionally.

Short-term exercise training improves cardiac function associated to a better antioxidant response and lower type 3 iodothyronine deiodinase activity after myocardial infarction.

AIMS: We assessed the effects of a short-term exercise training on cardiac function, oxidative stress markers, and type 3 iodothyronine deiodinase (D3) activity in cardiac tissue of spontaneously hypertensive rats (SHR) following experimental myocardial infarction (MI). METHODS: Twenty-four SHR (aged 3 months) were allocated to 4 groups: sham+sedentary, sham+trained, MI+sedentary and MI+trained. MI was performed by permanent ligation of the coronary artery. Exercise training (treadmill) started 96 hours after MI and lasted for 4 weeks (~60% maximum effort, 4x/week and 40 min/day). Cardiac function (echocardiography), thioredoxin reductase (TRx), total carbonyl levels, among other oxidative stress markers and D3 activity were measured. A Generalized Estimating Equation was used, followed by Bonferroni's test (p<0.05). RESULTS: MI resulted in an increase in left ventricular mass (p = 0.002) with decreased cardiac output (~22.0%, p = 0.047) and decreased ejection fraction (~41%, p = 0.008) as well as an increase in the carbonyl levels (p = 0.001) and D3 activity (~33%, p<0.001). Exercise training resulted in a decrease in left ventricular mass, restored cardiac output (~34%, p = 0.048) and ejection fraction (~20%, p = 0.040), increased TRx (~85%, p = 0.007) and reduced carbonyl levels (p<0.001) and D3 activity (p<0.001). CONCLUSIONS: Our short-term exercise training helped reverse the effects of MI on cardiac function. These benefits seem to derive from a more efficient antioxidant response and lower D3 activity in cardiac tissue.

Altered Gene Expression of Thyroid Hormone Transporters and Deiodinases in iPS MeCP2-Knockout Cells-Derived Neurons.

MeCP2 is an X-linked gene; its mutation causes Rett Syndrome (RTT), a severe neurodevelopmental disability that affects mainly girls. Acting as a transcription factor, the MeCP2 protein is able to regulate several hormone-related genes, such as the thyroid hormones (TH), which are known to play an important role in the development of the central nervous system (CNS). Although only a few studies have associated RTT and TH, TH deficit can lead to neurological deregulation by triggering functional deficiencies during adulthood. Here, we used human-induced pluripotent stem cell (iPSC) to generate MeCP2-knockout neuronal progenitor cells and adult neurons. Using this cellular model, we then investigated the expression of genes associated with TH homeostasis, such as the TH transporters (LAT1, LAT2, MCT8, MCT10, and OATP4A1) and deiodinases (DIO1, 2, and 3). Then, we treated the neural cells with THs and analyzed the expression of several genes related to neurodevelopment and functional maintenance. Our results showed that several TH-related genes, such as deiodinases, are altered in RTT samples when compared to WT cells. Moreover, the treatment of the neural cells with THs increased the amount of MAP2 and synapsin-1 expression in RTT cells. Our work provided evidences that TH homeostasis is compromised in RTT-derived neural cells, which could be an important factor to contribute to the imbalance in the neurodevelopmental phenotype presented in this syndrome and can lead us to better understand other neurodevelopmental diseases.

Propranolol inhibits myocardial infarction-induced brown adipose tissue D2 activation and maintains a low thyroid hormone state in rats

Considering the recognized role of thyroid hormones on the cardiovascular system during health and disease, we hypothesized that type 2 deiodinase (D2) activity, the main activation pathway of thyroxine (T4)-to-triiodothyronine (T3), could be an important site to modulate thyroid hormone status, which would then constitute a possible target for β-adrenergic blocking agents in a myocardial infarction (MI) model induced by left coronary occlusion in rats. Despite a sustained and dramatic fall in serum T4 concentrations (60-70%), the serum T3 concentration fell only transiently in the first week post-infarction (53%) and returned to control levels at 8 and 12 weeks after surgery compared to the Sham group (P<0.05). Brown adipose tissue (BAT) D2 activity (fmol T4·min-1·mg ptn-1) was significantly increased by approximately 77% in the 8th week and approximately 100% in the 12th week in the MI group compared to that of the Sham group (P<0.05). Beta-blocker treatment (0.5 g/L propranolol given in the drinking water) maintained a low T3 state in MI animals, dampening both BAT D2 activity (44% reduction) and serum T3 (66% reduction in serum T3) compared to that of the non-treated MI group 12 weeks after surgery (P<0.05). Propranolol improved cardiac function (assessed by echocardiogram) in the MI group compared to the non-treated MI group by 40 and 57%, 1 and 12 weeks after treatment, respectively (P<0.05). Our data suggested that the beta-adrenergic pathway may contribute to BAT D2 hyperactivity and T3 normalization after MI in rats. Propranolol treatment maintained low T3 state and improved cardiac function additionally.

Current concepts and challenges to unravel the role of iodothyronine deiodinases in human neoplasias.

Thyroid hormones (THs) are essential for the regulation of several metabolic processes and the energy consumption of the organism. Their action is exerted primarily through interaction with nuclear receptors controlling the transcription of thyroid hormone-responsive genes. Proper regulation of TH levels in different tissues is extremely important for the equilibrium between normal cellular proliferation and differentiation. The iodothyronine deiodinases types 1, 2 and 3 are key enzymes that perform activation and inactivation of THs, thus controlling TH homeostasis in a cell-specific manner. As THs seem to exert their effects in all hallmarks of the neoplastic process, dysregulation of deiodinases in the tumoral context can be critical to the neoplastic development. Here, we aim at reviewing the deiodinases expression in different neoplasias and exploit the mechanisms by which they play an essential role in human carcinogenesis. TH modulation by deiodinases and other classical pathways may represent important targets with the potential to oppose the neoplastic process.

Induction of Type 2 Iodothyronine Deiodinase After Status Epilepticus Modifies Hippocampal Gene Expression in Male Mice.

Status epilepticus (SE) is an abnormally prolonged seizure that results from either a failure of mechanisms that terminate seizures or from initiating mechanisms that inherently lead to prolonged seizures. Here we report that mice experiencing a 3 hours of SE caused by pilocarpine exhibit a rapid increase in expression of type 2 iodothyronine deiodinase gene (Dio2) and a decrease in the expression of type 3 iodothyronine deiodinase gene in hippocampus, amygdala and prefrontal cortex. Type 3 iodothyronine deiodinase in hippocampal sections was seen concentrated in the neuronal nuclei, typical of ischemic injury of the brain. An unbiased analysis of the hippocampal transcriptome of mice undergoing 3 hours of SE revealed a number of genes, including those involved with response to oxidative stress, cellular homeostasis, cell signaling, and mitochondrial structure. In contrast, in mice with targeted disruption of Dio2 in astrocytes (Astro D2KO mouse), the highly induced genes in the hippocampus were related to inflammation, apoptosis, and cell death. We propose that Dio2 induction caused by SE accelerates production of T3 in different areas of the central nervous system and modifies the hippocampal gene expression profile, affecting the balance between adaptive and maladaptive mechanisms.

Role of thyroid hormones in the neoplastic process: an overview.

Thyroid hormones (TH) are critical regulators of several physiological processes, which include development, differentiation and growth in virtually all tissues. In past decades, several studies have shown that changes in TH levels caused by thyroid dysfunction, disruption of deiodinases and/or thyroid hormone receptor (TR) expression in tumor cells, influence cell proliferation, differentiation, survival and invasion in a variety of neoplasms in a cell type-specific manner. The function of THs and TRs in neoplastic cell proliferation involves complex mechanisms that seem to be cell specific, exerting effects via genomic and nongenomic pathways, repressing or stimulating transcription factors, influencing angiogenesis and promoting invasiveness. Taken together, these observations indicate an important role of TH status in the pathogenesis and/or development of human neoplasia. Here, we aim to present an updated and comprehensive picture of the accumulated knowledge and the current understanding of the potential role of TH status on the different hallmarks of the neoplastic process.

Dissecting thyroid hormone transport and metabolism in dendritic cells.

We reported thyroid hormone (TH) receptor expression in murine dendritic cells (DCs) and 3,5,3'-triiodothyronine (T3)-dependent stimulation of DC maturation and ability to develop a Th1-type adaptive response. Moreover, an increased DC capacity to promote antigen-specific cytotoxic T-cell activity, exploited in a DC-based antitumor vaccination protocol, was revealed. However, putative effects of the main circulating TH, l-thyroxine (T4) and the mechanisms of TH transport and metabolism at DC level, crucial events for TH action at target cell level, were not known. Herein, we show that T4 did not reproduce those registered T3-dependent effects, finding that may reflect a homoeostatic control to prevent unspecific systemic activation of DCs. Besides, DCs express MCT10 and LAT2 TH transporters, and these cells mainly transport T3 with a favored involvement of MCT10 as its inhibition almost prevented T3 saturable uptake mechanism and reduced T3-induced IL-12 production. In turn, DCs express iodothyronine deiodonases type 2 and 3 (D2, D3) and exhibit both enzymatic activities with a prevalence towards TH inactivation. Moreover, T3 increased MCT10 and LAT2 expression and T3 efflux from DCs but not T3 uptake, whereas it induced a robust induction of D3 with a parallel slight reduction in D2. These findings disclose pivotal events involved in the mechanism of action of THs on DCs, providing valuable tools for manipulating the immunogenic potential of these cells. Furthermore, they broaden the knowledge of the TH mechanism of action at the immune system network.

Phosphodiesterase-7 inhibition affects accumbal and hypothalamic thyrotropin-releasing hormone expression, feeding and anxiety behavior of rats.

Thyrotropin-releasing hormone (TRH) has anorexigenic and anxiolytic functions when injected intraventricularly. Nucleus accumbens (NAcc) is a possible brain region involved, since it expresses proTRH. TRH from hypothalamic paraventricular nucleus (PVN) has a food intake-regulating role. TRHergic pathways of NAcc and PVN are implicated in anxiety and feeding. Both behaviors depend on cAMP and phosphorylated-cAMP response element binding protein (pCREB) intracellular levels. Intracellular levels of cAMP are controlled by the degrading activity of phosphodiesterases (PDEs). Since TRH transcription is activated by pCREB, a specific inhibitor of PDE7B may regulate TRH-induced effects on anxiety and feeding. We evaluated the effectiveness of an intra-accumbal and intraperitoneal (i.p.) administration of a PDE7 inhibitor (BRL-50481) on rats' anxiety-like behavior and food intake; also on TRH mRNA and protein expression in NAcc and PVN to define its mediating role on the PDE7 inhibitor-induced behavioral changes. Accumbal injection of 4µg/0.3µL of PDE7 inhibitor decreased rats' anxiety. The i.p. injection of 0.2mg/kg of the inhibitor was able to increase the PVN TRH mRNA expression and to decrease feeding but did not change animals' anxiety levels; in contrast, 2mg/kg b.w inhibitor enhanced accumbal TRH mRNA, induced anxiolysis with no change in food intake. PDE7 inhibitor induced anxiolytic and anorexigenic like behavior depending on the dose used. Results supported hypothalamic TRH mediated feeding-reduction effects, and accumbal TRH mediation of inhibitor-induced anxiolysis. Thus, an i.p dose of this inhibitor might be reducing anxiety with no change in feeding, which could be useful for obese patients.

New insights for male infertility revealed by alterations in spermatic function and differential testicular expression of thyroid-related genes.

The impact of thyroid hormone (TH) disorders on male reproductive biology has been a controversial issue for many years. Recently, we reported that hypothyroid male rats have a disruption of the seminiferous epithelium, which may compromise spermatogenesis. To improve the understanding of the reproductive pathogenesis of hypothyroidism and hyperthyroidism, male Wistar rats that developed these dysfunctions in adulthood were used as an experimental model. We evaluated the sperm production, reserves, transit time, morphology, and functionality (acrosome integrity, plasma membrane integrity, and mitochondrial activity), and the testicular expression of the TH receptors (Thra1 and Thra2, Thrb1, and Thrb2), deiodinases (Dio2 and Dio3), and the Mct8 transporter (Slc16a2) were assessed by reverse transcription followed by real-time quantitative PCR (RT-qPCR). The results were evaluated statistically by ANOVA and Tukey HSD test (P < 0.05). Hypothyroidism decreased the total and daily sperm productions and increased the sperm transit time through the epididymis, while the sperm functionality was reduced in both thyroid dysfunctions. Regarding the modulation of gene expression in the testis, hypothyroidism increased the expression of Thra1 and decreased the expression of Dio3, and hyperthyroidism increased the expression of Slc16a2. The observed alterations in spermatic production and function and in the expression of the TH receptor, deiodinase, and the TH transporter are suggestive of TH participation in spermatogenesis in adulthood.

Methylation differences and expression profiles of the caprine DIO3 gene.

DIO3 gene encoding type 3 iodothyronine deiodinase is an imprinted gene, located in the DLK1-DIO3 (delta-like 1 homolog-type 3 iodothyronine deiodinase) imprinted domain, and is potentially involved in degrading excessive amounts of thyroid hormone to protect embryogenesis. However, the underlying regulatory mechanism of the imprinted DIO3 gene expression during fetal and neonatal development in goats has not been elucidated. In this study, we explored the DNA methylation patterns of the caprine DIO3 intragenic CpG island and quantified gene expression level in six tissues from Chinese Nanjiang Yellow 3-day old kids. The expression of the DIO3 gene was determined using quantitative reverse transcription-polymerase chain reactions (qRT-PCRs), while the identification of methylation patterns was determined using bisulfite-sequencing PCRs. Modest, and non-significant (P > 0.05), methylation patterns were noted for the DIO3 CpG island methylation in the brain, heart, liver, kidney, lung, and longissimus dorsi tissues (ranging from 26.48 to 34.92%). The expression level of the DIO3 mRNA was significantly higher (P < 0.05) in the liver tissue than in the other five tissues. Pearson's correlation analysis revealed that there was no significant relationship between methylation and gene expression (P > 0.05), which indicated that the expression of the caprine DIO3 gene was likely modified by other regulatory elements. This study identified DNA methylation and expression patterns of the DIO3 gene in goats and provided insights into further regulatory mechanisms of expression and imprinting in the DLK1-DIO3 domain.

Underlying Mechanisms of Pituitary-Thyroid Axis Function Disruption by Chronic Iodine Excess in Rats.

BACKGROUND: Iodine is essential for thyroid hormone synthesis and is an important regulator of thyroid function. Chronic iodine deficiency leads to hypothyroidism, but iodine excess also impairs thyroid function causing hyperthyroidism, hypothyroidism, and/or thyroiditis. This study aimed to investigate the underlying mechanisms by which exposure to chronic iodine excess impairs pituitary-thyroid axis function. METHODS: Male Wistar rats were treated for two months with NaI (0.05% and 0.005%) or NaI+NaClO4 (0.05%) dissolved in drinking water. Hormone levels, gene expression, and thyroid morphology were analyzed later. RESULTS: NaI-treated rats presented high levels of iodine in urine, increased serum thyrotropin levels, slightly decreased serum thyroxine/triiodothyronine levels, and a decreased expression of the sodium-iodide symporter, thyrotropin receptor, and thyroperoxidase mRNA and protein, suggesting a primary thyroid dysfunction. In contrast, thyroglobulin and pendrin mRNA and protein content were increased. Kidney and liver deiodinase type 1 mRNA expression was decreased in iodine-treated rats. Morphological studies showed larger thyroid follicles with higher amounts of colloid and increased amounts of connective tissue in the thyroid of iodine-treated animals. All these effects were prevented when perchlorate treatment was combined with iodine excess. CONCLUSIONS: The present data reinforce and add novel findings about the disruption of thyroid gland function and the compensatory action of increased thyrotropin levels in iodine-exposed animals. Moreover, they draw attention to the fact that iodine intake should be carefully monitored, since both deficient and excessive ingestion of this trace element may induce pituitary-thyroid axis dysfunction.

Hypothalamus-Pituitary-Thyroid Axis.

The hypothalamus-pituitary-thyroid (HPT) axis determines the set point of thyroid hormone (TH) production. Hypothalamic thyrotropin-releasing hormone (TRH) stimulates the synthesis and secretion of pituitary thyrotropin (thyroid-stimulating hormone, TSH), which acts at the thyroid to stimulate all steps of TH biosynthesis and secretion. The THs thyroxine (T4) and triiodothyronine (T3) control the secretion of TRH and TSH by negative feedback to maintain physiological levels of the main hormones of the HPT axis. Reduction of circulating TH levels due to primary thyroid failure results in increased TRH and TSH production, whereas the opposite occurs when circulating THs are in excess. Other neural, humoral, and local factors modulate the HPT axis and, in specific situations, determine alterations in the physiological function of the axis. The roles of THs are vital to nervous system development, linear growth, energetic metabolism, and thermogenesis. THs also regulate the hepatic metabolism of nutrients, fluid balance and the cardiovascular system. In cells, TH actions are mediated mainly by nuclear TH receptors (210), which modify gene expression. T3 is the preferred ligand of THR, whereas T4, the serum concentration of which is 100-fold higher than that of T3, undergoes extra-thyroidal conversion to T3. This conversion is catalyzed by 5'-deiodinases (D1 and D2), which are TH-activating enzymes. T4 can also be inactivated by conversion to reverse T3, which has very low affinity for THR, by 5-deiodinase (D3). The regulation of deiodinases, particularly D2, and TH transporters at the cell membrane control T3 availability, which is fundamental for TH action. © 2016 American Physiological Society. Compr Physiol 6:1387-1428, 2016.

Differential responses of the somatotropic and thyroid axes to environmental temperature changes in the green iguana.

Growth hormone (GH), together with thyroid hormones (TH), regulates growth and development, and has critical effects on vertebrate metabolism. In ectotherms, these physiological processes are strongly influenced by environmental temperature. In reptiles, however, little is known about the direct influences of this factor on the somatotropic and thyroid axes. Therefore, the aim of this study was to describe the effects of both acute (48h) and chronic (2weeks) exposure to sub-optimal temperatures (25 and 18°C) upon somatotropic and thyroid axis function of the green iguana, in comparison to the control temperature (30-35°C). We found a significant increase in GH release (2.0-fold at 25°C and 1.9-fold at 18°C) and GH mRNA expression (up to 3.7-fold), mainly under chronic exposure conditions. The serum concentration of insulin-like growth factor-I (IGF-I) was significantly greater after chronic exposure (18.5±2.3 at 25°C; 15.92±3.4 at 18°C; vs. 9.3±1.21ng/ml at 35°C), while hepatic IGF-I mRNA expression increased up to 6.8-fold. Somatotropic axis may be regulated, under acute conditions, by thyrotropin-releasing hormone (TRH) that significantly increased its hypothalamic concentration (1.45 times) and mRNA expression (0.9-fold above control), respectively; and somatostatin (mRNA expression increased 1.0-1.2 times above control); and under chronic treatment, by pituitary adenylate cyclase-activating peptide (PACAP mRNA expression was increased from 0.4 to 0.6 times). Also, it was shown that, under control conditions, injection of TRH stimulated a significant increase in circulating GH. On the other hand, while there was a significant rise in the hypothalamic content of TRH and its mRNA expression, this hormone did not appear to influence the thyroid axis activity, which showed a severe diminution in all conditions of cold exposure, as indicated by the decreases in thyrotropin (TSH) mRNA expression (up to one-eight of the control), serum T4 (from 11.6±1.09 to 5.3±0.58ng/ml, after 2weeks at 18°C) and T3 (from 0.87±0.09 to 0.05±0.01ng/ml, under chronic conditions at 25°C), and Type-2 deiodinase (D2) activity (from 992.5±224 to 213.6±26.4fmolI(125)T4/mgh). The reduction in thyroid activity correlates with the down-regulation of metabolism as suggested by the decrease in the serum glucose and free fatty acid levels. These changes apparently were independent of a possible stress response, at least under acute exposure to both temperatures and in chronic treatment to 25°C, since serum corticosterone had no significant changes in these conditions, while at chronic 18°C exposure, a slight increase (0.38 times above control) was found. Thus, these data suggest that the reptilian somatotropic and thyroid axes have differential responses to cold exposure, and that GH and TRH may play important roles associated to adaptation mechanisms that support temperature acclimation in the green iguana.