Redefining the Role of Ornithine Aspartate and Vitamin E in Metabolic-Dysfunction-Associated Steatotic Liver Disease through Its Biochemical Properties.

It is known that the inflammation process leading to oxidative stress and thyroid hormone metabolism dysfunction is highly altered in metabolic dysfunction associated with steatotic liver disease (MASLD). This study aims to address the effect of ornithine aspartate (LOLA) and vitamin E (VitE) in improving these processes. Adult Sprague-Dawley rats were assigned to five groups and treated for 28 weeks: controls (n = 10) received a standard diet (for 28 weeks) plus gavage with distilled water (DW) from weeks 16 to 28. MASLD groups received a high-fat and choline-deficient diet for 28 weeks (MASLD group) and daily gavage with 200 mg/kg/day of LOLA, or twice a week with 150 mg of VitE from weeks 16-28. LOLA diminished collagen deposition (p = 0.006). The same treatment diminished carbonyl, TBARS, and sulfhydryl levels and GPx activity (p < 0.001). Type 3 deiodinase increased in the MASLD group, downregulating T3-controlled genes, which was corrected in the presence of LOLA. LOLA also promoted a near-normalization of complex II, SDH, and GDH activities (p < 0.001) and improved reticulum stress, with a reduction in GRP78 and HSPA9/GRP75 protein levels (p < 0.05). The enhanced energy production and metabolism of thyroid hormones, probably because of GSH replenishment provided by the L-glutamate portion of LOLA, opens a new therapeutic approach for MASLD.

A DIO2 missense mutation and its impact on fetal response to PRRSV infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) infection during late gestation substantially lowers fetal viability and survival. In a previous genome-wide association study, a single nucleotide polymorphism on chromosome 7 was significantly associated with probability of fetuses being viable in response to maternal PRRSV-2 infection at 21 days post maternal inoculation. The iodothyronine deiodinase 2 (DIO2) gene, located ~ 14 Kilobase downstream of this SNP, was selected as a priority candidate related to fetal susceptibility following maternal PRRSV-2 infection. Our objectives were to identify mutation(s) within the porcine DIO2 gene and to determine if they were associated with fetal outcomes after PRRSV-2 challenge. Sequencing of the DIO2, genotyping identified variants, and association of DIO2 genotypes with fetal phenotypes including DIO2 mRNA levels, viability, survival, viral loads, cortisol and thyroid hormone levels, and growth measurements were conducted. RESULTS: A missense variant (p.Asn91Ser) was identified in the parental populations from two independent PRRSV-2 challenge trials. This variant was further genotyped to determine association with fetal PRRS outcomes. DIO2 mRNA levels in fetal heart and kidney differed by the genotypes of Asn91Ser substitution with significantly greater DIO2 mRNA expression in heterozygotes compared with wild-type homozygotes (P < 0.001 for heart, P = 0.002 for kidney). While Asn91Ser did not significantly alter fetal viability and growth measurements, interaction effects of the variant with fetal sex or trial were identified for fetal viability or crown rump length, respectively. However, this mutation was not related to dysregulation of the hypothalamic-pituitary-adrenal and thyroid axis, indicated by no differences in circulating cortisol, T4, and T3 levels in fetuses of the opposing genotypes following PRRSV-2 infection. CONCLUSIONS: The present study suggests that a complex relationship among DIO2 genotype, DIO2 expression, fetal sex, and fetal viability may exist during the course of fetal PRRSV infection. Our study also proposes the increase in cortisol levels, indicative of fetal stress response, may lead to fetal complications, such as fetal compromise, fetal death, or premature farrowing, during PRRSV infection.

The Musashi-1-type 2 deiodinase pathway regulates astrocyte proliferation.

Thyroid hormone (TH) is a critical regulator of cellular function and cell fate. The circulating TH level is relatively stable, while tissue TH action fluctuates according to cell type-specific mechanisms. Here, we focused on identifying mechanisms that regulate TH action through the type 2 deiodinase (D2) in glial cells. Dio2 mRNA has an unusually long 3'UTR where we identified multiple putative MSI1 binding sites for Musashi-1 (MSI1), a highly conserved RNA-binding cell cycle regulator. Binding to these sites was confirmed through electrophoretic mobility shift assay. In H4 glioma cells, shRNA-mediated MSI1 knockdown increased endogenous D2 activity, whereas MSI1 overexpression in HEK293T cells decreased D2 expression. This latter effect could be prevented by the deletion of a 3.6 kb region of the 3'UTR of Dio2 mRNA containing MSI1 binding sites. MSI1 immunoreactivity was observed in 2 mouse Dio2-expressing cell types, that is, cortical astrocytes and hypothalamic tanycytes, establishing the anatomical basis for a potential in vivo interaction of Dio2 mRNA and MSl1. Indeed, increased D2 expression was observed in the cortex of mice lacking MSI1 protein. Furthermore, MSI1 knockdown-induced D2 expression slowed down cell proliferation by 56% in primary cultures of mouse cortical astrocytes, establishing the functionality of the MSI1-D2-T3 pathway. In summary, Dio2 mRNA is a target of MSI1 and the MSI1-D2-T3 pathway is a novel regulatory mechanism of astrocyte proliferation with the potential to regulate the pathogenesis of human glioblastoma.

Determination of Frequency of Type 2 Deiodinase Thr92Ala Polymorphism (rs225014) in 131I-treated Differentiated Thyroid Cancer Patients Undertaking L-thyroxine (L-T4) Suppression Therapy.

Introduction: Type 2 deiodinase (DIO2) enzyme plays a vital role in peripheral T4 to T3 conversion and in the negative feedback regulation of pituitary thyroid-stimulating hormone (TSH) secretion. Thr92Ala polymorphism (rs225014) is a common single-nucleotide polymorphism (SNP) that lowers DIO2 activity and is associated with diverse physiological disorders. Differentiated thyroid cancer (DTC) patients are given L-T4 therapy after total thyroidectomy and 131I treatment to suppress TSH levels. Aim: The aim of the study was to determine the frequency of rs225014 in DTC patients and to investigate its effect on the thyroid function tests (TFTs) and L-T4 dose required to suppress TSH levels. Materials and Methods: The study included a DTC patient group and a control group. TFTs were estimated by RIA/IRMA kits. Genomic DNA of all the subjects was screened for rs225014 SNP by polymerase chain reaction. Results: The frequency of Thr/Thr (wild type), Thr/Ala (heterozygous mutant), and Ala/Ala (homozygous mutant) genotypes in the DTC patients' group was 0.21, 0.52, and 0.27, respectively. T3 levels and T3/T4 ratio were significantly low in the Ala/Ala genotype in the DTC group indicating impaired DIO2 activity. L-T4 dose requirement to suppress TSH levels in the DTC patients harboring rs225014 SNP was not statistically different from the wild-type genotype. Conclusion: The SNP rs225014 was observed to be associated with T3 and T3/T4 ratio but not with the L-T4 dose in DTC harboring SNP suggesting the presence of a compensatory pathway to overcome DIO2 impairment. However, it is essential to study the genetic makeup of DTC patients showing reduced response to TSH suppression to enable quicker decision-making in the implementation of personalized L-T4 dose to prevent any adverse effects.

Prospective role of lusianthridin in attenuating cadmium-induced functional and cellular damage in rat thyroid.

The thyroid represents the most prevalent form of head and neck and endocrine cancer. The present investigation demonstrates the anticancer effects of Lusianthridin against cadmium (Cd)-induced thyroid cancer in rats. Swiss Wistar rats were utilized in this experimental study. Cd was employed to induce thyroid cancer, and the rats were divided into different groups, receiving oral administration of Lusianthridin (20 mg/kg) for 14 days. Thyroid parameters, deiodinase levels, hepatic parameters, lipid parameters, and antioxidant parameters were respectively estimated. The mRNA expression was assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR). Lusianthridin significantly (P < 0.001) improved protein levels, T4, T3, free iodine in urine, and suppressed the level of TSH. Lusianthridin significantly (P < 0.001) enhanced the levels of FT3, FT4, and decreased the level of rT3. Lusianthridin significantly (P < 0.001) reduced the levels of D1, D2, D3, and enhanced the levels of hepatic parameters like AST, ALT. Lusianthridin remarkably (P < 0.001) altered the levels of lipid parameters such as LDL, total cholesterol, HDL, and triglycerides; antioxidant parameters viz., MDA, GSH, CAT, and SOD. Lusianthridin significantly altered the mRNA expression of Bcl-2, Bax, MEK1, ERK1, ERK2, p-eIf2α, GRP78, eIf2α, and GRP94. The results clearly state that Lusianthridin exhibits protective effects against thyroid cancer.

The selenoenzyme type I iodothyronine deiodinase: a new tumor suppressor in ovarian cancer.

The selenoenzyme type I iodothyronine deiodinase (DIO1) catalyzes removal of iodine atoms from thyroid hormones. Although DIO1 action is reported to be disturbed in several malignancies, no work has been conducted in high-grade serous ovarian carcinoma (HGSOC), the most lethal gynecologic cancer. We studied DIO1 expression in HGSOC patients [The Cancer Genome Atlas (TCGA) data and tumor tissues], human cell lines (ES-2 and Kuramochi), normal Chinese hamster ovarian cells (CHO-K1), and normal human fallopian tube cells (FT282 and FT109). To study its functional role, DIO1 was overexpressed, inhibited [by propylthiouracil (PTU)], or knocked down (KD), and cell count, proliferation, apoptosis, cell viability, and proteomics analysis were performed. Lower DIO1 levels were observed in HGSOC compared to normal cells and tissues. TCGA analyses confirmed that low DIO1 mRNA expression correlated with worse survival and therapy resistance in patients. Silencing or inhibiting the enzyme led to enhanced ovarian cancer proliferation, while an opposite effect was shown following DIO1 ectopic expression. Proteomics analysis in DIO1-KD cells revealed global changes in proteins that facilitate tumor metabolism and progression. In conclusion, DIO1 expression and ovarian cancer progression are inversely correlated, highlighting a tumor suppressive role for this enzyme and its potential use as a biomarker in this disease.

Cardiac recovery from pressure overload is not altered by thyroid hormone status in old mice.

Introduction: Thyroid hormones (THs) are known to have various effects on the cardiovascular system. However, the impact of TH levels on preexisting cardiac diseases is still unclear. Pressure overload due to arterial hypertension or aortic stenosis and aging are major risk factors for the development of structural and functional abnormalities and subsequent heart failure. Here, we assessed the sensitivity to altered TH levels in aged mice with maladaptive cardiac hypertrophy and cardiac dysfunction induced by transverse aortic constriction (TAC). Methods: Mice at the age of 12 months underwent TAC and received T4 or anti-thyroid medication in drinking water over the course of 4 weeks after induction of left ventricular pressure overload. Results: T4 excess or deprivation in older mice had no or only very little impact on cardiac function (fractional shortening), cardiac remodeling (cardiac wall thickness, heart weight, cardiomyocyte size, apoptosis, and interstitial fibrosis), and mortality. This is surprising because T4 excess or deprivation had significantly changed the outcome after TAC in young 8-week-old mice. Comparing the gene expression of deiodinases (Dio) 2 and 3 and TH receptor alpha (TRα) 1 and the dominant-negative acting isoform TRα2 between young and aged mice revealed that aged mice exhibited a higher expression of TRα2 and Dio3, while expression of Dio2 was reduced compared with young mice. These changes in Dio2 and 3 expressions might lead to reduced TH availability in the hearts of 12-month-old mice accompanied by reduced TRα action due to higher TRα2. Discussion: In summary, our study shows that low and high TH availability have little impact on cardiac function and remodeling in older mice with preexisting pressure-induced cardiac damage. This observation seems to be the result of an altered expression of deiodinases and TRα isoforms, thus suggesting that even though cardiovascular risk is increasing with age, the response to TH stress may be dampened in certain conditions.

A combination of silymarin and garlic extract enhances thyroid hormone activation and body metabolism in orally intoxicated male rats with atrazine: Impact on hepatic iodothyronine deiodinase type 1.

Atrazine is a widely applied herbicide to improve crop yield and maintain general health. It has been reported to impair thyroid function and architecture in experimental animals. Alterations in thyroid hormones disrupt normal body function and metabolism. Silymarin, a hepatoprotective flavonolignan, was found to improve thyroid function and body metabolism. Additionally, garlic displays several protective effects on body organs. Therefore, this study explored the prophylactic impact of natural compounds comprising silymarin and garlic extract on disrupted thyroid function, hepatic iodothyronine deiodinase type 1, and metabolic parameters in atrazine-intoxicated male rats. We found that daily pre- and co-treatment of atrazine-intoxicated male rats with silymarin (100 mg/kg, p.o) and/or garlic extract (10 mg/kg, p.o) significantly improved thyroid activation and hepatic functionality as evidenced by the re-establishment of T3, T3/T4, and TSH values as well as ALT and AST activities. Interestingly, individual or concurrent supplementation of the atrazine group with silymarin and garlic extract prevented the down-regulation in hepatic iodothyronine deiodinase type 1. These effects were coupled with the repletion of serum and hepatic antioxidants and the amelioration of lipid peroxidation. In addition, current natural products markedly alleviated weight gain, dyslipidemia, hyperglycemia, glucose intolerance, and insulin resistance. Notably, a cocktail of silymarin and garlic extract exerted superior protection against atrazine-triggered deterioration of thyroid, hepatic, and metabolic functioning to individual treatments. Present findings pinpoint the prophylactic and synergistic influence of silymarin and garlic extract combinatorial regimen on thyroid activation and body metabolism via enhancing antioxidant potential, maintaining hepatic function, and iodothyronine deiodinase type 1.

Mice lacking DIO3 exhibit sex-specific alterations in circadian patterns of corticosterone and gene expression in metabolic tissues.

Disruption of circadian rhythms is associated with neurological, endocrine and metabolic pathologies. We have recently shown that mice lacking functional type 3 deiodinase (DIO3), the enzyme that clears thyroid hormones, exhibit a phase shift in locomotor activity, suggesting altered circadian rhythm. To better understand the physiological and molecular basis of this phenotype, we used Dio3+/+ and Dio3-/- mice of both sexes at different zeitgeber times (ZTs) and analyzed corticosterone and thyroxine (T4) levels, hypothalamic, hepatic, and adipose tissue expression of clock genes, as well as genes involved in the thyroid hormone action or physiology of liver and adipose tissues. Wild type mice exhibited sexually dimorphic circadian patterns of genes controlling thyroid hormone action, including Dio3. Dio3-/- mice exhibited altered hypothalamic expression of several clock genes at ZT12, but did not disrupt the overall circadian profile. Expression of clock genes in peripheral tissues was not disrupted by Dio3 deficiency. However, Dio3 loss in liver and adipose tissues disrupted circadian profiles of genes that determine tissue thyroid hormone action and physiology. We also observed circadian-specific changes in serum T4 and corticosterone as a result of DIO3 deficiency. The circadian alterations manifested sexual dimorphism. Most notable, the time curve of serum corticosterone was flattened in Dio3-/- females. We conclude that Dio3 exhibits circadian variations, influencing the circadian rhythmicity of thyroid hormone action and physiology in liver and adipose tissues in a sex-specific manner. Circadian disruptions in tissue physiology may then contribute to the metabolic phenotypes of DIO3-deficient mice.

Urine Se concentration poorly predicts plasma Se concentration at sub-district scales in Zimbabwe, limiting its value as a biomarker of population Se status.

Introduction: The current study investigated the value of urine selenium (Se) concentration as a biomarker of population Se status in rural sub-Saharan Africa. Method: Urine and plasma Se concentrations were measured among children aged 6-59 months (n = 608) and women of reproductive age (WRA, n = 781) living in rural Zimbabwe (Murehwa, Shamva, and Mutasa districts) and participating in a pilot national micronutrient survey. Selenium concentrations were measured by inductively coupled plasma-mass spectrometry (ICP-MS), and urine concentrations were corrected for hydration status. Results: The median (Q1, Q3) urine Se concentrations were 8.4 µg/L (5.3, 13.5) and 10.5 µg/L (6.5, 15.2) in children and WRA, respectively. There was moderate evidence for a relationship between urine Se concentration and plasma Se concentration in children (p = 0.0236) and WRA (p = < 0.0001), but the relationship had poor predictive value. Using previously defined thresholds for optimal activity of iodothyronine deiodinase (IDI), there was an association between deficiency when indicated by plasma Se concentrations and urine Se concentrations among WRA, but not among children. Discussion: Urine Se concentration poorly predicted plasma Se concentration at sub-district scales in Zimbabwe, limiting its value as a biomarker of population Se status in this context. Further research is warranted at wider spatial scales to determine the value of urine Se as a biomarker when there is greater heterogeneity in Se exposure.

Effect of DIO2 Gene Polymorphism on Thyroid Hormone Levels and Its Correlation with the Severity of Schizophrenia in a Pakistani Population.

Low levels of triiodothyronine (T3) in the brain lead to increased dopamine receptor sensitivity, potentially resulting in schizophrenia. Iodothyronine deiodinase 2 (DIO2) is the only enzyme which converts tetraiodothyronine (T4) to T3 in the brain. DIO2 polymorphism of rs225014 results in the expression of non-functioning DIO2. Therefore, this study aimed to investigate the association of rs255014 with schizophrenia and its impact on thyroid hormone levels. This study included 150 schizophrenia cases and 150 controls. DNA was extracted from blood and subjected to PCR and amplicon sequencing. Serum thyroid profiles were determined using chemiluminescent magnetic microparticle immunoassay. Statistical analyses involved independent sample t-tests, Chi-square, and Pearson's correlation tests. The results revealed a higher frequency of the reference genotype (TT) in controls compared to cases (p < 0.05). However, rs225014 did not influence serum thyroid levels or the severity of schizophrenia (p > 0.05). Interestingly, control subjects exhibited significantly higher T3 levels (p < 0.001) than cases. Regardless of the genotype (TT or CC), the control group had higher mean T3 levels than the corresponding case group (p < 0.05). In conclusion, rs225014 is associated with schizophrenia and has no effect on serum thyroid hormone levels.

Neural thyroid hormone metabolism integrates seasonal changes in environmental temperature with the neuroendocrine reproductive axis.

We asked if environmental temperature alters thyroid hormone metabolism within the hypothalamus, thereby providing a neuroendocrine mechanism by which temperature could be integrated with photoperiod to regulate seasonal rhythms. We used immunohistochemistry to assess the effects of low-temperature winter dormancy at 4 °C or 12 °C on thyroid-stimulating hormone (TSH) within the infundibulum of the pituitary as well as deiodinase 2 (Dio2) and 3 (Dio3) within the hypothalamus of red-sided garter snakes (Thamnophis sirtalis). Both the duration and, in males, magnitude of low-temperature dormancy altered deiodinase immunoreactivity within the hypothalamus, increasing the area of Dio2-immunoreactivity in males and females and decreasing the number of Dio3-immunoreactive cells in males after 8-16 weeks. Reciprocal changes in Dio2/3 favor the accumulation of triiodothyronine within the hypothalamus. Whether TSH mediates these effects requires further study, as significant changes in TSH-immunoreactive cell number were not observed. Temporal changes in deiodinase immunoreactivity coincided with an increase in the proportion of males exhibiting courtship behavior as well as changes in the temporal pattern of courtship behavior after emergence. Our findings mirror those of previous studies, in which males require low-temperature exposure for at least 8 weeks before significant changes in gonadotropin-releasing hormone immunoreactivity and sex steroid hormones are observed. Collectively, these data provide evidence that the neuroendocrine pathway regulating the reproductive axis via thyroid hormone metabolism is capable of transducing temperature information. Because all vertebrates can potentially use temperature as a supplementary cue, these results are broadly applicable to understanding how environment-organism interactions mediate seasonally adaptive responses.

Regulation of Thyroid Hormone Gatekeepers by Thyrotropin in Tanycytes.

Background: Tanycytes are specialized glial cells within the mediobasal hypothalamus that have multiple functions, including hormone sensing and regulation of hypophysiotropic hormone secretion. There are ongoing discussions about the role of tanycytes in regulating the supply of hypothalamic thyroid hormones (THs) through the expression of TH transporters (Slc16a2, Slco1c1) and deiodinases (Dio2, Dio3). In this study, we investigated the potential feedback effect of thyrotropin (TSH) on the transcription of these gatekeeper genes on tanycytes. Methods: We analyzed the changes in the expression of TH-gatekeeper genes, in TSH-stimulated primary tanycytes, using quantitative polymerase chain reaction (qPCR). We also used RNAScope® in brain slices to further reveal the local distribution of the transcripts. In addition, we blocked intracellular pathways and used small-interfering RNA (siRNA) to elucidate differences in the regulation of the gatekeeper genes. Results: TSH elevated messenger RNA (mRNA) levels of Slco1c1, Dio2, and Dio3 in tanycytes, while Slc16a2 was mostly unaffected. Blockade and knockdown of the TSH receptor (TSHR) and antagonization of cAMP response element-binding protein (CREB) clearly abolished the increased expression induced by TSH, indicating PKA-dependent regulation through the TSHR. The TSH-dependent expression of Dio3 and Slco1c1 was also regulated by protein kinase C (PKC), and in case of Dio3, also by extracellular signal-regulated kinase (ERK) activity. Importantly, these gene regulations were specifically found in different subpopulations of tanycytes. Conclusions: This study demonstrates that TSH induces transcriptional regulation of TH-gatekeeper genes in tanycytes through the Tshr/Gαq/PKC pathway, in parallel to the Tshr/Gαs/PKA/CREB pathway. These differential actions of TSH on tanycytic subpopulations appear to be important for coordinating the supply of TH to the hypothalamus and aid its functions.

Demonstration of the Formation of a Selenocysteine Selenenic Acid through Hydrolysis of a Selenocysteine Selenenyl Iodide Utilizing a Protective Molecular Cradle.

Selenocysteine selenenic acids (Sec-SeOHs) and selenocysteine selenenyl iodides (Sec-SeIs) have long been recognized as crucial intermediates in the catalytic cycle of glutathione peroxidase (GPx) and iodothyronine deiodinase (Dio), respectively. However, the observation of these reactive species remained elusive until our recent study, where we successfully stabilized Sec-SeOHs and Sec-SeIs using a protective molecular cradle. Here, we report the first demonstration of the chemical transformation from a Sec-SeI to a Sec-SeOH through alkaline hydrolysis. A stable Sec-SeI derived from a selenocysteine methyl ester was synthesized using the protective cradle, and its structure was determined by crystallographic analysis. The alkaline hydrolysis of the Sec-SeI at -50 °C yielded the corresponding Sec-SeOH in an 89% NMR yield, the formation of which was further confirmed by its reaction with dimedone. The facile and nearly quantitative conversion of the Sec-SeI to the Sec-SeOH not only validates the potential involvement of this process in the catalytic mechanism of Dio, but also highlights its utility as a method for producing a Sec-SeOH.

Thyroid hormone transport and metabolism are disturbed in the placental villi of miscarriage.

BACKGROUND: It has been long known that thyroid hormone regulates placental villi development, which is associated with the occurrence of miscarriage. However, whether abnormal thyroid hormone metabolism and transport in placental villi are involved in miscarriage is still to be verified. METHODS: Placental villi of elective terminations of pregnancies (ETPs) and miscarriage were collected. Proliferative activity and apoptosis of villi trophoblasts and angiogenesis were detected by TUNEL and immunochemistry. The expressions of thyroid hormone receptors (THRs), transthyretin (TTR), monocarboxylate transporter 8 (MCT8), organic anion transporting polypeptides 1A1 (OATP1A1), deiodinase 2 (Dio2) and Dio3 were examined by RT-PCR, Western blot, immunohistochemistry and immunofluorescence. JEG3 cell was treated with iopanoic acid (IOP), an inhibitor of Dio2 activity, the expressions of Dio2, placenta growth factor (PLGF) and sFlt1 were detected by RT-PCR and Western blot. RESULTS: Cell proliferation was suppressed and apoptosis was increased in placental villi cytotrophoblasts of miscarriage. CD34+ vessel number and vascular endothelial growth factor (VEGF) protein abundance were decreased in miscarriage. In miscarriage group, the gene expression of Dio2, Dio3, TTR and THRα, but not THRß, MCT8 and OATP1A1, were downregulated. The protein abundances of TTR and THRα were downregulated in miscarriage group, but not THRß. The protein abundance of Dio2 in miscarriage villi was decreased compared with that in ETP. In JEG3 cells, the gene expression of PLGF was decreased and the expression of sFlt1 was increased in IOP treatment; The protein abundance of Dio2 was downregulated but the gene expression of Dio2 was unaffected in IOP treatment. CONCLUSION: Thyroid hormone transport and metabolism in miscarriage were disturbed and may impaired angiogenesis of placental villi, which was associated with the occurrence of miscarriage.

Designing a combined liothyronine (LT3), L- thyroxine (LT4) trial in symptomatic hypothyroid subjects on LT4 - the importance of patient selection, choice of LT3 and trial design.

Approximately 10%-15% of subjects with hypothyroidism on L-thyroxine (LT4) alone have persistent symptoms affecting their quality of life (QoL). Although the cause is unclear, there is evidence that "tissue T3 lack" may be responsible. If so, combining liothyronine (LT3) with LT4 would be helpful. However, randomized controlled trials (RCT), have not established greater efficacy for the LT3 + LT4 combination in these subjects than for LT4 alone. While the trial design may have been responsible, the use of unphysiological, short-acting LT3 preparations and non-thyroid-specific patient-reported outcome measures (PROMs) may have contributed. We recommend attention to the following aspects of trial design for future RCTs of LT3 + LT4 compared to LT4 alone: (a) Subject selection-(i) measurable symptoms (disadvantages should be recognized); (ii) using a validated thyroid specific PROM such as ThyPRO39 or the Composite scale derived from it; (iii) those taking over 1.2 µg/day or 100 µg/day (for pragmatic reasons) of LT4 defining a population likely without intrinsic thyroid activity who depend on exogenous LT4; (iv) recruiting a preponderance of subjects with autoimmune thyroiditis increasing generalisability; and (v) those with a high symptom load with a greater response to combination therapy e.g. those with the deiodinase 2 polymorphism. (b) The use of physiological LT3 preparations producing pharmacokinetic similarities to T3 profiles in unaffected subjects: two long-acting LT3 preparations are currently available and must be tested in phase 2b/3 RCTs. (c) The superiority of a crossover design in limiting numbers and costs while maintaining statistical power and ensuring that all subjects experienced the investigative medication.

Decreased hepatic thyroid hormone signaling in systemic and liver-specific but not brain-specific accelerated aging due to DNA repair deficiency in mice.

Background: Thyroid hormone signaling is essential for development, metabolism, and response to stress but declines during aging, the cause of which is unknown. DNA damage accumulating with time is a main cause of aging, driving many age-related diseases. Previous studies in normal and premature aging mice, due to defective DNA repair, indicated reduced hepatic thyroid hormone signaling accompanied by decreased type 1 deiodinase (DIO1) and increased DIO3 activities. We investigated whether aging-related changes in deiodinase activity are driven by systemic signals or represent cell- or organ-autonomous changes. Methods: We quantified liver and plasma thyroid hormone concentrations, deiodinase activities and expression of T3-responsive genes in mice with a global, liver-specific and for comparison brain-specific inactivation of Xpg, one of the endonucleases critically involved in multiple DNA repair pathways. Results: Both in global and liver-specific Xpg knockout mice, hepatic DIO1 activity was decreased. Interestingly, hepatic DIO3 activity was increased in global, but not in liver-specific Xpg mutants. Selective Xpg deficiency and premature aging in the brain did not affect liver or systemic thyroid signaling. Concomitant with DIO1 inhibition, Xpg -/- and Alb-Xpg mice displayed reduced thyroid hormone-related gene expression changes, correlating with markers of liver damage and cellular senescence. Conclusions: Our findings suggest that DIO1 activity during aging is predominantly modified in a tissue-autonomous manner driven by organ/cell-intrinsic accumulating DNA damage. The increase in hepatic DIO3 activity during aging largely depends on systemic signals, possibly reflecting the presence of circulating cells rather than activity in hepatocytes.

Sustained Pituitary T3 Production Explains the T4-mediated TSH Feedback Mechanism.

The regulation of thyroid activity and thyroid hormone (TH) secretion is based on feedback mechanisms that involve the anterior pituitary TSH and medial basal hypothalamus TSH-releasing hormone. Plasma T3 levels can be "sensed" directly by the anterior pituitary and medial basal hypothalamus; plasma T4 levels require local conversion of T4 to T3, which is mediated by the type 2 deiodinase (D2). To study D2-mediated T4 to T3 conversion and T3 production in the anterior pituitary gland, we used mouse pituitary explants incubated with 125I-T4 for 48 hours to measure T3 production at different concentrations of free T4. The results were compared with cultures of D1- or D2-expressing cells, as well as freshly isolated mouse tissue. These studies revealed a unique regulation of the D2 pathway in the anterior pituitary gland, distinct from that observed in nonpituitary tissues. In the anterior pituitary, increasing T4 levels reduced D2 activity slightly but caused a direct increase in T3 production. However, the same changes in T4 levels decreased T3 production in human HSkM cells and murine C2C12 cells (both skeletal muscle) and mouse bone marrow tissue, which reached zero at 50 pM free T4. In contrast, the increase in T4 levels caused the pig kidney LLC-PK1 cells and kidney fragments to proportionally increase T3 production. These findings have important implications for both physiology and clinical practice because they clarify the mechanism by which fluctuations in plasma T4 levels are transduced in the anterior pituitary gland to mediate the TSH feedback mechanism.

Deiodinase Types 1 and 3 and Proinflammatory Cytokine Values May Discriminate Depressive Disorder Patients from Healthy Controls.

INTRODUCTION: Depressive disorders are multifactorial diseases in that a variety of factors may play a role in their etiology, including inflammation and abnormalities in the thyroid hormone (TH) metabolism and levels. The purpose of this study was to evaluate iodothyronine deiodinases (DIOs) and DIO-interacting cytokines as possible biomarkers in the diagnosis of depressive disorders. METHODS: This study enrolled 73 patients diagnosed with recurrent depressive disorder (rDD) and 54 controls. The expressions of DIO1, DIO2, DIO3, IL1B, IL6, TNFA, and IFNG genes, encoding three types of DIOs (1, 2, and 3), interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, were assessed using the polymerase chain reaction in blood cells and an enzymatic immunoassay method in serum. The levels of examined molecules between patients and controls were compared, and correlations and diagnostic values were evaluated. RESULTS: Lower levels of DIO2 and higher levels of IL1B, IL6, and TNFA were found in patients compared to controls. The protein concentrations of DIO1 and DIO2 were lower, while that of DIO3 was higher, in patients than in controls. Serum IL-1ß, IL-6, and TNF-α were also higher in patients than in controls. The area under the curve (AUC) of the IL-1ß, IL-6, DIO1, and DIO3 proteins was >0.7 for discriminating patients with rDD from controls. CONCLUSIONS: The expressions of genes for DIO2, IL-1ß, IL-6, and TNF-α may have a role in the estimation of processes present in depressive disorders. We can cautiously claim that DIO1 and DIO3 and pivotal cytokines, mainly IL-1ß and IL-6, may play a role in depression diagnosis, and further studies are suggested to explain the exact role of these molecules in larger samples with more precise methods.

Gene polymorphisms and thyroid hormone signaling: implication for the treatment of hypothyroidism.

INTRODUCTION: Mutations and single nucleotide polymorphisms (SNPs) in the genes encoding the network of proteins involved in thyroid hormone signaling (TH) may have implications for the effectiveness of the treatment of hypothyroidism with LT4. It is conceivable that loss-of-function mutations or SNPs impair the ability of LT4 to be activated to T3, reach its targets, and ultimately resolve symptoms of hypothyroidism. Some of these patients do benefit from therapy containing LT4 and LT3. METHODS: Here, we reviewed the PubMed and examined gene mutations and SNPs in the TH cellular transporters, deiodinases, and TH receptors, along with their impact on TH signaling, and potential clinical implications. RESULTS: In some mechanisms, such as the Thr92Ala-DIO2 SNP, there is a compelling rationale for reduced T4 to T3 activation that limits the effectiveness of LT4 to restore euthyroidism. In other mechanisms, a potential case can be made but more studies with a larger number of individuals are needed. DISCUSSION/CONCLUSION: Understanding the clinical impact of the genetic makeup of LT4-treated patients may help in the preemptive identification of those individuals that would benefit from therapy containing LT3.