Comparison of in vitro thyroxine (T4) metabolism between Wistar rat and human hepatocyte cultures.

In vitro assays remain relatively new in exploring human relevance of liver, in particular nuclear receptor-mediated perturbations of the hypothalamus-pituitary-thyroid axis seen in rodents, mainly in the rat. Consistent with in vivo data, we confirm that thyroid hormone thyroxine metabolism was 9 times higher in primary rat hepatocytes (PRH) than in primary human hepatocytes (PHH) cultured in a 2D sandwich (2Dsw) configuration. In addition, thyroxine glucuronide (T4-G) was by far the major metabolite formed in both species (99.1% in PRH and 69.7% in PHH) followed by thyroxine sulfate (T4-S, 0.7% in PRH and 18.1% in PHH) and triiodothyronine/reverse triiodothyronine (T3/rT3, 0.2% in PRH and 12.2% in PHH). After a 7-day daily exposure to orphan receptor-mediated liver inducers, T4 metabolism was strongly increased in PRH, almost exclusively through increased T4-G formation. These results were consistent with the inductions of glucuronosyltransferase Ugt2b1 and canalicular transporter Mrp2. PHH also responded to activation of the three nuclear receptors, with mainly induction of glucuronosyltransferase UGT1A1 and canalicular transporter MRP2. Despite this, T4 disappearance rate and secreted T4 metabolites were only slightly increased in PHH. Overall, our data highlight that cryopreserved hepatocytes in 2Dsw culture allowing long-term exposure and species comparison are of major interest in improving liver-mediated human safety assessment.

The Effect of Long-Term Inorganic Iodine on Intrathyroidal Iodothyronine Content and Gene Expression in Mice with Graves' Hyperthyroidism.

Background: The main molecular mechanism underlying acute suppression of iodine organification in normal thyroids after an excessive iodine load, that is, the Wolff-Chaikoff effect, is assumed to be suppression of iodine oxidation and iodothyronine synthesis. However, the mechanism underlying chronic antithyroid action of inorganic iodine in Graves' disease is not fully understood. Using a mouse model of Graves' hyperthyroidism, we examined changes in iodothyronine content and gene expression profiles in the thyroid glands after inorganic iodine loading. Materials and Methods: Graves' hyperthyroidism was induced and maintained in BALB/c mice by repeated immunizations of recombinant adenovirus expressing the human thyrotropin (TSH) receptor A-subunit. Hyperthyroid mice were left untreated (GD-C; n = 8) or treated with inorganic iodine for 12 weeks (GD-NaI; n = 8). We used unimmunized BALB/c mice as a control group (n = 10). In each mouse, serum thyroxine (T4) levels were measured with enzyme-linked immunosorbent assay (ELISA) at 4-week intervals. The intrathyroidal iodothyronine content and gene expression levels were, respectively, evaluated by mass spectrometry and RNA sequencing (RNA-seq) at the end of the experimental period. Results: Serum T4 levels in the GD-C group remained higher than in the control group, whereas those in the GD-NaI group declined to normal levels during the experimental period. Intrathyroidal triiodothyronine (T3), reverse T3 (rT3), and T4 contents in the GD-C group were higher than the control group, and rT3 and T4 were further increased in the GD-NaI group. The observed alterations in iodothyronine levels in the thyroid and sera may be explained by altered expression levels of genes for iodothyronine biosynthetic molecules, their transporter, and deiodinases. Conclusion: In this mouse model of hyperthyroidism, higher intrathyroidal accumulation of T4 and reduced gene expression data of iodothyronine transporters in the GD-NaI group suggest that chronic antithyroid action of iodine in Graves' disease involves suppression of hormone secretion.

Advantages of monitoring rT3 and dividing LT3 dose in the treatment of consumptive hypothyroidism associated with infantile hepatic hemangioma.

OBJECTIVES: In consumptive hypothyroidism associated with infantile hepatic hemangiomas (IHH), elevated reverse triiodothyronine (rT3) is known due to elevated D3. This report shows that rT3 is a new indicator of IHH progression and that three divided doses of LT3 per day were more effective than a single dose. CASE PRESENTATION: A 23 day-old boy was diagnosed with diffuse IHH and severe hypothyroidism with high rT3. Propranolol and LT4 were administered. Hemangiomas gradually diminished and rT3 decreased, but the thyroid-stimulating hormone remained elevated, and free triiodothyronine (fT3) did not normalize after 2 weeks of treatment. Liothyronine (LT3) was started as a single dose and then divided into three doses after 1 week, which stabilized thyroid function. CONCLUSIONS: rT3 levels were less variable and decreased in conjunction with tumor shrinkage; thus, rT3 is an indicator of therapeutic outcomes for IHH. LT3 administered in divided doses aided in managing IHH-associated hypothyroidism.

Molecular Basis for the Remarkably Different Gas-Phase Behavior of Deprotonated Thyroid Hormones Triiodothyronine (T3) and Reverse Triiodothyronine (rT3): A Clue for Their Discrimination?

Thyroid hormones are biologically active small molecules responsible for growth and development regulation, basal metabolic rate, and lipid and carbohydrate metabolism. Liquid chromatography mass spectrometry (LC-MS) can be used to quantify thyroid hormones blood level with high speed and selectivity, aiming to improve the diagnosis and treatment of the severe pathological conditions in which they are implicated, i.e., hypo- and hyperthyroidism. In this work, the gas-phase behavior of the isomeric thyroid hormones triiodothyronine (T3) and reverse triiodothyronine (rT3) in their deprotonated form was studied at a molecular level using MS-based techniques. Previously reported collision-induced dissociation experiments yielded distinct spectra despite the high structural similarity of the two compounds, suggesting different charge sites to be responsible. Infrared multiple photon dissociation spectroscopy on [T3-H]- and [rT3-H]- was performed, and the results were interpreted using DFT and MP2 calculations, assessing the prevalence of T3 in the carboxylate form and rT3 as a phenolate isomer. The different deprotonation sites of the two isomers were also found to drive their ion-mobility behavior. In fact, [T3-H]- and [rT3-H]- were successfully separated. Drift times were correlated with collisional cross section values of 209 and 215 Å2 for [T3-H]- and [rT3-H]-, respectively. Calculations suggested the charge site to be the main parameter involved in the different mobilities of the two anions. Finally, bare [T3-H]- and [rT3-H]- were made to react with neutral acetylacetone and trifluoroacetic acid, confirming rT3 to be more acidic than T3 in agreement with the calculated gas-phase acidities of T3 and rT3 equal to 1345 and 1326 kJ mol-1, respectively.

Physiological Role and Use of Thyroid Hormone Metabolites - Potential Utility in COVID-19 Patients.

Thyroxine and triiodothyronine (T3) are classical thyroid hormones and with relatively well-understood actions. In contrast, the physiological role of thyroid hormone metabolites, also circulating in the blood, is less well characterized. These molecules, namely, reverse triiodothyronine, 3,5-diiodothyronine, 3-iodothyronamine, tetraiodoacetic acid and triiodoacetic acid, mediate both agonistic (thyromimetic) and antagonistic actions additional to the effects of the classical thyroid hormones. Here, we provide an overview of the main factors influencing thyroid hormone action, and then go on to describe the main effects of the metabolites and their potential use in medicine. One section addresses thyroid hormone levels in corona virus disease 19 (COVID-19). It appears that i) the more potently-acting molecules T3 and triiodoacetic acid have shorter half-lives than the less potent antagonists 3-iodothyronamine and tetraiodoacetic acid; ii) reverse T3 and 3,5-diiodothyronine may serve as indicators for metabolic dysregulation and disease, and iii) Nanotetrac may be a promising candidate for treating cancer, and resmetirom and VK2809 for steatohepatitis. Further, the use of L-T3 in the treatment of severely ill COVID-19 patients is critically discussed.

In Vitro Characterization of Human, Mouse, and Zebrafish MCT8 Orthologues.

Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause MCT8 deficiency, characterized by severe intellectual and motor disability and abnormal serum thyroid function tests. Various Mct8 knock-out mouse models as well as mct8 knock-out and knockdown zebrafish models are used as a disease model for MCT8 deficiency. Although important for model eligibility, little is known about the functional characteristics of the MCT8 orthologues in these species. Therefore, we here compared the functional characteristics of mouse (mm) MCT8 and zebrafish (dr) Mct8 to human (hs) MCT8. Methods: We performed extensive transport studies in COS-1 and JEG-3 cells transiently transfected with hsMCT8, drMct8, and mmMCT8. Protein expression levels and subcellular localization were assessed by immunoblotting, surface biotinylation, and immunocytochemistry. Sequence alignment and structural modeling were used to interpret functional differences between the orthologues. Results: hsMCT8, drMct8, and mmMCT8 all facilitated the uptake and efflux of 3,3'-diiodothyronine (3,3'-T2), rT3, triiodothyronine (T3), and thyroxine (T4), although the initial uptake rates of drMct8 were 1.5-4.0-fold higher than for hsMCT8 and mmMCT8. drMct8 exhibited 3-50-fold lower apparent IC50 values than hsMCT8 and mmMCT8 for all tested substrates, and substrate preference of drMct8 (3,3'-T2, T3 > T4 > rT3) differed from hsMCT8 and mmMCT8 (T3 > T4 > rT3, 3,3'-T2). Compared with hsMCT8 and mmMCT8, cis-inhibition studies showed that T3 uptake by drMct8 was inhibited at a lower concentration and by a broader spectrum of TH metabolites. Total and cell surface expression levels of drMct8 and hsMCT8 were equal and both significantly exceeded those of mmMCT8. Structural modeling located most non-conserved residues outside the substrate pore, except for H192 in hsMCT8, which is replaced by a glutamine in drMct8. However, a H192Q substituent of hsMCT8 did not alter its transporter characteristics. Conclusion: Our studies substantiate the eligibility of mice and zebrafish models for human MCT8 deficiency. However, differences in the intrinsic transporter properties of MCT8 orthologues may exist, which should be realized when comparing MCT8 deficiency in different in vivo models. Moreover, our findings may indicate that the protein domains outside the substrate channel may play a role in substrate selection and protein stability.

Action of Reverse T3 on Cancer Cells.

Background: Reverse T3 (rT3; 3,3',5'-triiodo-L-thyronine) is widely regarded as an inactive naturally occurring analog of thyroid hormone. rT3 is known to bind to the thyroid hormone analog receptor on plasma membrane integrin αvß3. This integrin is generously expressed by tumor cells and is the initiation site for the stimulation by L-thyroxine (T4) at physiological free concentrations on cancer cell proliferation. Results: In the present studies, we show that rT3 caused increases of proliferation in vitro of 50% to 80% (P < 0.05-0.001) of human breast cancer and glioblastoma cells. Conclusion: rT3 may be a host factor supporting cancer growth.

Reverse T3 interacts with αvß3 integrin receptor and restores enzyme activities in the hippocampus of hypothyroid developing rats: Insight on signaling mechanisms.

In the present study we provide evidence that 3,3',5'-triiodothyronine (reverse T3, rT3) restores neurochemical parameters induced by congenital hypothyroidism in rat hippocampus. Congenital hypothyroidism was induced by adding 0.05% propylthiouracil in the drinking water from gestation day 8 and continually up to lactation day 15. In the in vivo rT3 exposure, hypothyroid 12-day old pups were daily injected with rT3 (50 ng/kg body weight) or saline until day 14. In the ex vivo rT3 treatment, hippocampal slices from 15-day-old hypothyroid pups were incubated for 30 min with or without rT3 (1 nM). We found that ex vivo and/or in vivo exposure to rT3 failed in restoring the decreased 14C-glutamate uptake; however, restored the phosphorylation of glial fibrillary acidic protein (GFAP), 45Ca2+ influx, aspartate transaminase (AST), glutamine synthetase (GS) and gamma-glutamate transferase (GGT) activities, as well as glutathione (GSH) levels in hypothyroid hippocampus. In addition, rT3 improved 14C-2-deoxy-D-glucose uptake and lactate dehydrogenase (LDH) activity. Receptor agonists/antagonists (RGD peptide and AP-5), kinase inhibitors of p38MAPK, ERK1/2, CaMKII, PKA (SB239063, PD98059, KN93 and H89, respectively), L-type voltage-dependent calcium channel blocker (nifedipine) and intracellular calcium chelator (BAPTA-AM) were used to determine the mechanisms of the nongenomic rT3 action on GGT activity. Using molecular docking analysis, we found rT3 interaction with αvß3 integrin receptors, nongenomically activating signaling pathways (PKA, CaMKII, p38MAPK) that restored GGT activity. We provide evidence that rT3 is an active TH metabolite and our results represent an important contribution to elucidate the nonclassical mechanism of action of this metabolite in hypothyroidism.

Thyroid hormone treatment activates protective pathways in both in vivo and in vitro models of neuronal injury.

Thyroid hormone plays an important role in brain development and adult brain function, and may influence neuronal recovery after Traumatic Brain Injury (TBI). We utilized both animal and cell culture models to determine the effects of thyroid hormone treatment, post TBI or during hypoxia, on genes important for neuronal survival and neurogenesis. We show that TBI in rats is associated with a reduction in serum thyroxine (T4) and triiodothyronine (T3). A single dose of levothyroxine (T4), one hour after injury, increased serum T4 and normalized serum T3 levels. Expression of genes important for thyroid hormone action in the brain, MCT8 and Type 2 deiodinase (Dio2) mRNA, diminished after injury, but were partially restored with T4 treatment. mRNA from the Type 3 deiodinase (Dio3) gene, which inactivates T4 to reverse T3 (rT3), was induced 2.7 fold by TBI, and further stimulated 6.7-fold by T4 treatment. T4 treatment significantly increased the expression of mRNA from Bcl2, VEGFA, Sox2 and neurotrophin, genes important for neuronal survival and recovery. The cortex, compared to the hippocampus and cerebellum, sustained the greatest injury and had the most significant change in gene expression as a result of injury and the greatest response to T4 treatment. We utilized hypoxia to study the effect of neuronal injury in vitro. Neuroblastoma cells were exposed to reduced oxygen tension, 0.2%, and were compared to cells grown at control oxygen levels of 21%. T3 treatment significantly increased hypoxia inducible factor (HIF)-2α protein, but not HIF-1α. In a hypoxia time course exposure, expression of hypoxia-mediated genes (VEGF, Enolase, HIF2α, c-Jun) peaked at least 8 h earlier with T3-treatment, compared to cells grown without T3. The early induction of these genes may promote cellular growth after injury. After hypoxic injury, T3 induced mRNA expression of the genes, KLF9 and hairless, important for T3-mediated brain function. The findings from both in vitro and in vivo studies support a role of thyroid hormone in activating pathways important for neuronal protection and promotion of neuronal recovery after injury.

Serum Thyroid Function, Mortality and Disability in Advanced Old Age: The Newcastle 85+ Study.

CONTEXT: Perturbations in thyroid function are common in older individuals but their significance in the very old is not fully understood. OBJECTIVE: This study sought to determine whether thyroid hormone status and variation of thyroid hormones within the reference range correlated with mortality and disability in a cohort of 85-year-olds. DESIGN: A cohort of 85-year-old individuals were assessed in their own homes (community or institutional care) for health status and thyroid function, and followed for mortality and disability for up to 9 years. SETTING AND PARTICIPANTS: Six hundred and forty-three 85-year-olds registered with participating general practices in Newcastle and North Tyneside, United Kingdom. MAIN OUTCOMES: All-cause mortality, cardiovascular mortality, and disability according to thyroid disease status and baseline thyroid hormone parameters (serum TSH, FT4, FT3, and rT3). Models were adjusted for age, sex, education, body mass index, smoking, and disease count. RESULTS: After adjustment for age and sex, all-cause mortality was associated with baseline serum rT3 and FT3 (both P < .001), but not FT4 or TSH. After additional adjustment for potential confounders, only rT3 remained significantly associated with mortality (P = .001). Baseline serum TSH and rT3 predicted future disability trajectories in men and women, respectively. CONCLUSIONS: Our study is reassuring that individuals age 85 y with both subclinical hypothyroidism and subclinical hyperthyroidism do not have a significantly worse survival over 9 years than their euthyroid peers. However, thyroid function tests did predict disability, with higher serum TSH levels predicting better outcomes. These data strengthen the argument for routine use of age-specific thyroid function reference ranges.

Type 1 5'-deiodinase activity is inhibited by oxidative stress and restored by alpha-lipoic acid in HepG2 cells.

3,3',5-triiodothyronine (T3) is largely generated from thyroxine (T4) by the catalysis of deiodinases in peripheral tissues. Emerging evidences have indicated its broad participation in regulating various metabolic process via protecting tissues from oxidative stress and improving cellular antioxidant capacity. However, the potential correlation between the oxidative stress and conversion of T4 to T3 is still unclear. In the present study, the effects of T3 and T4 on redox homeostasis in HepG2 cells pre-treated with H2O2 was investigated. It revealed that T3 significantly rescued the apoptotic cell death, consistent with an upregulation of cell antioxidant ability and reduction of ROS accumulation while T4 did not. Afterwards, we examined the enzyme activity and mRNA expression of type 1 5'-deiodianse (DIO1), T3 and rT3 level and found that H2O2 reduced both DIO1 activity and expression in a dose-dependent manner, which consequently declined T3 and rT3 generation. Alpha-lipoic acid (LA) treatment notably restored DIO1 activity, T3 and rT3 level, as well as transcriptional abnormalities of inflammation-associated genes. It suggests that oxidative stress may reduce DIO1 activity by an indirect way like activating cellular inflammatory responses. All these results indicate that the oxidative stress downregulates the conversion of T4 to T3 through DIO1 function in HepG2 cells.

Relation of thyroid hormone abnormalities with subclinical inflammatory activity in patients with type 1 and type 2 diabetes mellitus.

Thyroid hormone (TH) abnormalities are common in patients with diabetes mellitus (DM). These thyroid hormone abnormalities have been associated with inflammatory activity in several conditions but this link remains unclear in DM. We assessed the influence of subclinical inflammation in TH metabolism in euthyroid diabetic patients. Cross-sectional study involving 258 subjects divided in 4 groups: 70 patients with T2DM and 55 patients with T1DM and two control groups of 70 and 63 non-diabetic individuals, respectively. Groups were paired by age, sex, and body mass index (BMI). We evaluated the association between clinical and hormonal variables [thyrotropin, reverse T3 (rT3), total and free thyroxine (T4), and triiodothyronine (T3)] with the inflammation markers C-reactive protein (hs-CRP), serum amyloid A (SAA), and interleukin-6 (IL-6). Serum T3 and free T3 were lower in patients with diabetes (all P < 0.001) compared to the control groups. Interleukin-6 showed positive correlations with rT3 in both groups (P < 0.05). IL-6 was independently associated to FT3/rT3 (B = -0.193; 95% CI -0.31; -0.076; P = 0.002) and FT4/rT3 (B = -0.107; 95% CI -0.207; -0.006; P = 0.039) in the T1DM group. In the T2DM group, SAA (B = 0.18; 95% CI 0.089; 0.271; P < 0.001) and hs-CRP (B = -0.069; 95% CI -0.132; -0.007; P = 0.03) predicted FT3 levels. SAA (B = -0.16; 95% CI -0.26; -0.061; P = 0.002) and IL6 (B = 0.123; 95% CI 0.005; 0.241; P = 0.041) were related to FT4/FT3. In DM, differences in TH levels compared to non-diabetic individuals were related to increased subclinical inflammatory activity and BMI. Altered deiodinase activity was probably involved. These findings were independent of sex, age, BMI, and HbA1c levels.

Environmental heat stress modulates thyroid status and its response to repeated endotoxin challenge in steers.

The objective of this study was to evaluate in cattle, the effects of acute exposure to a heat stress (HS) environment on the status of the pituitary (thyrotropin, TSH)-thyroid (thyroxine, T4)-peripheral tissue T4 deiodination (type 1 5'-deiodinase [D1]; triiodothyronine [T3]; reverse-triiodothyronine [rT3]) axis, and the further response of this pituitary-thyroid-peripheral tissue axis (PTTA) to perturbation caused by the induction of the proinflammatory innate immune state provoked by the administration of gram-negative bacteria endotoxin (lipopolysaccharide [LPS]). Ten steers (318 ± 49 kg body weight) housed in controlled environment chambers were subjected to either a thermoneutral (TN: constant 19°C) or HS temperature conditions (cyclical daily temperatures: 32.2°C-40.0°C) for a total period of 9 d. To minimize the effects of altered plane of nutrition due to HS, steers in TN were pair-fed to animals in HS conditions. Steers received 2 LPS challenges 3 d apart (LPS1 and LPS2; 0.2 µg/kg body weight, intravenously, Escherichia coli 055:B5) with the first challenge administered on day 4 relative to the start of the environmental conditioning. Jugular blood samples were collected at 0, 1, 2, 4, 7, and 24 h relative to the start of each LPS challenge. Plasma TSH, T4, T3, and rT3 were measured by radioimmunoassay. Liver D1 activity was measured in biopsy samples collected before the LPS1 (0 h) and 24 h after LPS2. Before the start of LPS1, HS decreased (P < 0.01 vs TN) plasma TSH (40%), T4 (45.4%), and T3 (25.9%), but did not affect rT3 concentrations. In TN steers, the LPS1 challenge decreased (P < 0.01 vs 0 h) plasma concentrations of TSH between 1 and 7 h and T4 and T3 at 7 and 24 h. In HS steers, plasma TSH concentrations were decreased at 2 h only (P < 0.05), whereas plasma T3 was decreased at 7 and 24 h (P < 0.01). Whereas plasma T4 concentrations were already depressed in HS steers at 0 h, LPS1 did not further affect the levels. Plasma rT3 concentrations were increased in all steers at 4, 7, and 24 h after LPS1 (P < 0.01). The patterns of concentration change of T4, T3, and rT3 during LPS2 mirrored those observed in LPS1; the responses in plasma TSH were of smaller magnitude than those incurred after LPS1. The LPS challenges reduced (P < 0.01) hepatic activity of D1 in all animals but no differences were observed between steers subjected to TN or HS environment. The data are consistent with the concept that acute exposure of cattle to a HS environment results in the depression of the pituitary and thyroid components of the PTTA, whereas a normal capacity to generate T3 from T4 in the liver is preserved. The data also suggest that LPS challenge further suppresses all components of the PTTA including liver T3 generation, and these PTTA perturbations are more pronounced in steers that encounter a HS exposure.

In vitro transdifferentiation of human cultured CD34+ stem cells into oligodendrocyte precursors using thyroid hormones.

The extent of myelination on the axon promotes transmission of impulses in the neural network, any disturbances in this process results in the neurodegenerative condition. Transplantation of oligodendrocyte precursors that supports in the regeneration of axons through myelination is an important step in the restoration of damaged neurons. Therefore, in the present study, the differentiation of human CD34+ stem cells into oligodendrocytes was carried out. The pure human CD34+ culture developed from the stem cells obtained from a peripheral blood of a donor were subjected to oligodendrocyte differentiation medium (ODM). The ODM at a concentration of 40ng/ml thyroxine, 40ng/ml 3,3',5-tri-iodo-thyronine showed distinct morphological changes from day 6 to 9 with cells exhibiting conspicuous stellate morphology and extensive foot processes. The real-time PCR analysis showed prominent expression of Olig2, CNPase, PDGFRα and PLP1/DM20 in the differentiated cells confirming the formed cells are oligodendrocyte precursors. The expression of these genes increased from days 6 to 9 corresponding to the morphological changes observed with almost no expression of GFAP+ cells. The distinct CNPase activity was observed in these differentiated cells compared to normal CD34+ stem cells correlating with results of real-time PCR conclusively explains the development of oligodendrocytes from human CD34+ stem cells.

Glucagon-like peptide-1 stimulates type 3 iodothyronine deiodinase expression in a mouse insulinoma cell line.

AIMS: The pathophysiological roles of thyroid hormones in glucose metabolism remain uncertain. Type 3 iodothyronine deiodinase (D3) converts thyroxine (T4) and 3,5,3'-triiodothyronine (T3) to 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine (T2), respectively, inactivating thyroid hormones in a cell-specific fashion. In the present study, we identified D3 expression in MIN6 cells derived from a mouse insulinoma cell line and examined the mechanisms regulating D3 expression in these cells. MAIN METHODS: We characterized D3 activity using HPLC analysis, and examined the effect of GLP-1 or exendin-4 on D3 expression and cAMP accumulation in MIN6 cells. We also measured insulin secretion from MIN6 cells exposed to GLP-1 and T3. KEY FINDINGS: We identified enzyme activity that catalyzes the conversion of T3 to T2 in MIN6 cells, which showed characteristics compatible with those for D3. D3 mRNA was identified in these cells using RT-PCR analysis. Forskolin rapidly stimulated D3 mRNA and D3 activity. Glucagon-like peptide-1 (GLP-1) increased D3 expression in a dose-dependent manner, and this effect was inhibited by the protein kinase A (PKA) inhibitor H-89. Exendin-4, a GLP-1 receptor agonist, also stimulated D3 expression in MIN6 cells. These results suggest that a cAMP-PKA-mediated pathway participates in GLP-1-stimulated D3 expression in MIN6 cells. Furthermore, GLP-1 stimulated insulin secretion was suppressed by the addition of T3 in MIN6 cells. SIGNIFICANCE: Our findings indicate that GLP-1 regulates intracellular T3 concentration in pancreatic ß cells via a cAMP-PKA-D3-mediated pathway that may also regulate ß-cell function.

Rapid responses to reverse T3 hormone in immature rat Sertoli cells: calcium uptake and exocytosis mediated by integrin.

There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T3 (rT3) action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT3 are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT3 was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT3-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions). Therefore, our findings suggest that calcium uptake stimulated by rT3 may be mediated by integrin αvß3. In addition, it was demonstrated that calcium uptake stimulated by rT3 is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT3 also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT3 modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology.

Heredity and lifestyle in the determination of between-subject variation in thyroid hormone levels in euthyroid men.

OBJECTIVE: Variation in thyroid hormone (TH) concentrations between subjects is greater than in a single subject over a prolonged period of time, suggesting an individual set point for thyroid function. We have previously shown that TH levels within normal range are associated with clinical indices such as bone mass, BMI, and heart rate. The aim of this study on young men was therefore to gain insight into the determinants of variation in TH levels among healthy subjects. METHODS: Healthy male siblings (n=941, 25-45 years) were recruited in a cross-sectional, population-based study; a history or treatment of thyroid disease and thyroid auto-immunity were exclusion criteria. A complete assessment of TH status was performed (TSH, free thyroxine (FT4), free triiodothyronine (FT3), thyroperoxidase, and thyroglobulin antibodies, reverse T3 (rT3), thyroid-binding globulin (TBG), and urinary iodine levels). Genotyping was performed by TaqMan and KASP (KBiosciences) genotyping assays. RESULTS: (F)T4, rT3, and TBG had heritability estimates between 80 and 90%. Estimates were lower for (F)T3 (60%) and lowest for TSH (49%). Significant associations were observed between different single-nucleotide polymorphisms (SNPs) in the thyroid pathway and TSH, FT4, ratio FT3:FT4, and rT3. Nevertheless, these SNPs only explain a limited part of the heredity. As to age and lifestyle-related factors, (F)T3 was negatively related to age and education level, positively to smoking and BMI (all P<0.0001) but not substantially to urinary iodine concentrations. Smoking was also negatively related to TSH and positively to FT4. CONCLUSION: Both genetic and lifestyle-related factors play a role in determining between-subject variation in TH levels in euthyroid young men, although genetic factors seem most important.

Deiodination in cancer growth: the role of type III deiodinase.

Thyroid hormone (TH) is a pleiotropic agent that has widespread biological functions, i.e., it controls cellular growth, tissue development and homeostasis and neoplastic transformation. Suitable TH levels are critical for the development of various types of tissues and are essential for the regulation of metabolic processes throughout life. The serum concentrations of TH affect its biological activity. Moreover, at tissue level, TH action is regulated by the expression and activity of deiodinases, i.e., the enzymes that mediate the metabolic pathways by activating and/or inactivating TH. The type I and II deiodinases (D1 and D2) initiate TH action by converting thyroxine (T4) into the active TH form (T3), whereas type III deiodinase (D3) mediates the local attenuation of TH by converting T4 and T3 into the inactive metabolites rT3 and T2, respectively. The deiodinase system is a potent mechanism of pre-receptoral control of TH action; it is often altered in such pathological conditions as cancer. D3 is widely expressed in embryonic tissues and in placenta, where it blocks excessive maternal-to-fetal transfer of TH. In contrast, during late neonatal and adult life, D3 is expressed mainly in the central nervous system and skin. Interestingly, D3 expression is re-activated in various types of human cancers. Here we review recent evidence that D3 expression plays a crucial role in human carcinogenesis, and speculate as to its complex role in the regulation of cell proliferation in several neoplastic contexts. It is conceivable that the local modulation of TH action via deiodinases is a powerful molecular tool to manipulate the intracellular TH status, thus influencing the growth and maintenance of selected hormone-dependent cancers.

Síndrome do eutireoidiano doente em pacientes com diabetes mellitus tipo 2: correlação com marcadores inflamatórios, controle glicêmico e relação com doença cardiovascular / Euthyroid sick syndrome in patients with type 2 diabetes mellitus: correlation between inflammatory markers, glycemic control and relationship with cardiovascular disease

A síndrome do eutireoidiano doente (SED) é uma entidade caracterizada pela queda das concentrações sanguíneas de triiodotironina nas formas total e livre e aumento da forma reversa. Ocorre principalmente em pacientes portadores de doenças graves e agudas, particularmente dentre aqueles internados em unidade de terapia intensiva. Há descrição desta síndrome em portadores de Diabetes Mellitus, particularmente sob controle glicêmico inadequado. Objetivos: Avaliar as alterações dos hormônios tireoidianos em portadores de DM sob cuidado ambulatorial e a correlação entre concentrações de hormônios tireoidianos e controle glicêmico, presença de complicações crônicas (neuropatia, nefropatia, retinopatia) e marcadores de inflamação sistêmica subclínica, bem como sua relação com presença de eventos cardiovasculares. Metodologia: Estudo transversal avaliando 52 pacientes com diabetes tipo 2 e 52 indivíduos sem diabetes, entre 40 e 75 anos de idade, pareados por sexo, idade e índice de massa corporal. Avaliaram-se dados clínicos e antropométricos, concentrações séricas de hormônios tireoidianos e proteína C reativa, bem como exames laboratoriais que refletem o perfil lipídico e controle glicêmico. Resultados: Cerca de 73% dos pacientes com diabetes e 40% dos indivíduos sem DM apresentaram concentrações séricas diminuídas de T3 total; 25% dos pacientes e apenas 2% dos indivíduos sem DM apresentaram concentrações diminuídas de T3 livre. As concentrações séricas de T3 total (p<0,001), T3 livre (p<0,001) e T4 total (p=0,006) estavam diminuídas em comparação aos de indivíduos sem diabetes. As concentrações de T3 reverso não apresentaram diferença entre os dois grupos. Pacientes com diabetes apresentaram T4 livre mais elevado (p=0,033).

The non-thyroidal illness is an entity characterized by reduced serum levels of total and free triiodothyronine and a rise in its reverse form. It occurs mainly in critically ill patients. There are descriptions of this syndrome in patients with Diabetes Mellitus, especially those under inadequate glycemic control. Objectives: Evaluate the abnormalities in thyroid hormone levels in individuals with diabetes under standard outpatient care and the correlation of thyroid hormone levels with glycemic control, presence of chronic complications (neuropathy, nephropathy and retinopathy) and subclinical systemic inflammation, as well as its relation with the presence of previous cardiovascular events. Methodology: Cross sectional study involving 52 patients with type 2 diabetes and 52 individuals without the diabetes, between 40 and 75 years of age paired by age, gender and body mass index. We evaluated clinical and anthropometric data, serum levels of thyroid hormones and Creactive protein, as well as laboratory parameters that reflect the lipid profile and glycemic control. Results: Approximately 73% of the patients with diabetes and 40% of individuals without diabetes presented reduced serum levels of total T3. Nearly 25% of the patients and only 2% of the individuals without diabetes presented reduced levels of free T3. The levels of total T3 (p<0.001), free T3 (p<0.001) and total T4 (p=0.006) were lower in patients with diabetes compared with those without diabetes. The levels of reverse T3 did not present any difference between both groups. Patients with diabetes presented higher levels of free T4 (p=0.033). The levels of reverse T3 were significantly different only when comparing individuals with previous cardiovascular events with those without this characteristic (p=0.002 for patients with diabetes and p=0.037 for individuals without diabetes). The prevalence of cardiovascular disease was 25%.

3,5,3'-triiodothyronine (T3) stimulates cell proliferation through the activation of the PI3K/Akt pathway and reactive oxygen species (ROS) production in chick embryo hepatocytes.

Thyroid hormones (THs) have a wide variety of essential roles in vertebrates, ranging from the regulation of key metabolic processes to cell proliferation and apoptosis. The classical mechanism of action of THs is genomic; 3,5,3'-triiodothyronine (T3) binds to specific nuclear receptors (TRs) and modifies the expression of specific genes. Recently, a new category of mechanisms, termed nongenomic, has been discovered for T3. These mechanisms include, among others, the rapid activation of signal transduction pathways, such as PI3K/Akt and MAPK, which eventually lead to cell proliferation. These effects are mediated in some cell types by a plasma membrane receptor, identified as integrin αvß3, and in other cell types by cytoplasmic TRß1. The aim of this work was to analyze the effect of T3 on the cell growth of chick embryo hepatocytes at two different stages of development, 14 and 19 days, and to determine the activation of the signal transduction pathways, focusing on the potential involvement of a plasma membrane receptor and the possible participation of PI3K/Akt and reactive oxygen species (ROS). Our results clearly show that T3 stimulates cell proliferation at both stages of development through the activation of the PI3K/Akt pathway and the production of small amounts of ROS, which operate as effective second messengers. Moreover, we prove that these effects are not initiated at the plasma membrane receptor for T3.