Is There a Link between Thyroid Peroxidase Gene Promoter Polymorphisms and Autoimmune Thyroiditis in the Polish Population?

(1) Autoimmune thyroiditis (AIT) is the most common cause of primary hypothyroidism and one of the most frequent organ-specific autoimmune diseases. Its pathogenesis is polygenic and still requires further research. The aim of the study was to assess, for the first time in the Caucasian population, the role of selected TPO gene promoter polymorphisms (rs2071399 G/A, rs2071400C/T, rs2071402 A/G, and rs2071403 A/G) in the development of AIT. A total of 237 patients diagnosed with AIT and 130 healthy controls were genotyped for four TPO gene polymorphisms, and the results were statistically analyzed to check for the role of these polymorphisms. There were no significant differences in the genotype and allele frequencies of the studied TPO gene promoter polymorphisms between patients and controls (p > 0.05). The haplotype distribution (rs2071400-rs2071402-rs2071403) between the two studied groups was similar for the most common variants (CGA, CAG, TGG). Only a rare haplotype (CGG) occurred more frequently among patients compared to controls (p = 0.04). The studied TPO gene promoter polymorphisms did not show an association with susceptibility to AIT in the Caucasian Polish population, contrary to the results in Japanese patients.

A regulatory element associated to NAFLD in the promoter of DIO1 controls LDL-C, HDL-C and triglycerides in hepatic cells.

BACKGROUND: Genome-wide association studies (GWAS) have identified genetic variants linked to fat metabolism and related traits, but rarely pinpoint causative variants. This limitation arises from GWAS not considering functional implications of noncoding variants that can affect transcription factor binding and potentially regulate gene expression. The aim of this study is to investigate a candidate noncoding functional variant within a genetic locus flagged by a GWAS SNP associated with non-alcoholic fatty liver disease (NAFLD), a condition characterized by liver fat accumulation in non-alcohol consumers. METHODS: CRISPR-Cas9 gene editing in HepG2 cells was used to modify the regulatory element containing the candidate functional variant linked to NAFLD. Global gene expression in mutant cells was assessed through RT-qPCR and targeted transcriptomics. A phenotypic assay measured lipid droplet accumulation in the CRISPR-Cas9 mutants. RESULTS: The candidate functional variant, rs2294510, closely linked to the NAFLD-associated GWAS SNP rs11206226, resided in a regulatory element within the DIO1 gene's promoter region. Altering this element resulted in changes in transcription factor binding sites and differential expression of candidate target genes like DIO1, TMEM59, DHCR24, and LDLRAD1, potentially influencing the NAFLD phenotype. Mutant HepG2 cells exhibited increased lipid accumulation, a hallmark of NAFLD, along with reduced LDL-C, HDL-C and elevated triglycerides. CONCLUSIONS: This comprehensive approach, that combines genome editing, transcriptomics, and phenotypic assays identified the DIO1 promoter region as a potential enhancer. Its activity could regulate multiple genes involved in the NAFLD phenotype or contribute to defining a polygenic risk score for enhanced risk assessment in NAFLD patients.

A Computational Approach: The Functional Effects of Thyroid Peroxidase Variants in Thyroid Cancer and Genetic Disorders.

PURPOSE: Thyroid peroxidase (TPO) is essential for the synthesis of thyroid hormones. However, specific mutations render TPO antigenic and prone to autoimmune attacks leading to thyroid cancer, TPO deficiency, and congenital hypothyroidism (CH). Despite technological advancement, most experimental procedures cannot quickly identify the genetic causes of CH nor detect thyroid cancer in the early stages. METHODS: We performed saturated computational mutagenesis to calculate the folding energy changes (∆∆G) caused by missense mutations and analyzed the mutations involved in post-translational modifications (PTMs). RESULTS: Our results showed that the functional important missense mutations occurred in the heme peroxidase domain. Through computational saturation mutagenesis, we identified the TPO mutations in G393 and G348 affecting protein stability and PTMs. Our folding energy calculations revealed that seven of nine somatic thyroid cancer mutations destabilized TPO. CONCLUSION: These findings highlight the impact of these specific mutations on TPO stability, linking them to thyroid cancer and other genetic thyroid-related disorders. Our results show that computational mutagenesis of proteins provides a quick insight into rare mutations causing Mendelian disorders and cancers in humans.

IL-10-TG/TPO-T4 axis, the target of bis (2-ethylhexyl) tetrabromophthalate on thyroid function imbalance.

Bis (2-ethylhexyl) tetrabromophthalate (TBPH) is a new type of brominated flame retardant. Some studies suggest that TBPH exposure may be associated with thyroid damage. However, there is a paucity of research on the authentic exposure-related effects and molecular mechanisms in animals or cells. In this study, we used male Sprague-Dawley (SD) rats and the Nthy ori3-1 cell line (the human thyroid follicular epithelial cell) to explore the potential effects of TBPH (5, 50, 500 mg/kg and 1, 10, 100 nM) on the thyroid. The genes and their proteins of cytokines and thyroid-specific proteins, thyroglobulin (TG), thyroid peroxidase (TPO), and sodium iodide cotransporter (NIS) were examined to investigate the possible mechanisms. At the end of the experiment, it was found that 50 and 500 mg/kg TBPH could increase the levels of total thyroxine (TT4) and free thyroxine (FT4) significantly. The messenger RNAs (mRNAs) of Tg, Tpo, Interleukin-6 (Il6), and Interleukin-10 (Il10) in the thyroid tissues from the rats treated with 500 mg/kg were enhanced clearly. Meanwhile, the mRNAs of TG, TPO, IL6, and IL10 were elevated in Nthy ori3-1 cells treated with 100 nM TBPH as well. The mRNAs of TG and TPO were elevated after the knockdown of IL6. To our surprise, after the knockdown of IL10 or the treatment of anti-IL-10-receptor (anti-IL-10-R) antibody, the mRNAs of TG and TPO were significantly reduced, and the effects of TBPH were diminished. In conclusion, our results suggested that the IL-10-IL-10R-TG/TPO-T4 axis is one important target of TBPH in the thyroid.

Effects of Perfluorooctane Sulfonate on Cerebellar Cells via Inhibition of Type 2 Iodothyronine Deiodinase Activity.

Perfluorooctane sulfonate (PFOS) has been used in a wide variety of industrial and commercial products. The adverse effects of PFOS on the developing brain are becoming of a great concern. However, the molecular mechanisms of PFOS on brain development have not yet been clarified. We investigated the effect of early-life exposure to PFOS on brain development and the mechanism involved. We investigated the change in thyroid hormone (TH)-induced dendrite arborization of Purkinje cells in the primary culture of newborn rat cerebellum. We further examined the mechanism of PFOS on TH signaling by reporter gene assay, quantitative RT-PCR, and type 2 iodothyronine deiodinase (D2) assay. As low as 10-7 M PFOS suppressed thyroxine (T4)-, but not triiodothyronine (T3)-induced dendrite arborization of Purkinje cells. Reporter gene assay showed that PFOS did not affect TRα1- and TRß1-mediated transcription in CV-1 cells. RT-PCR showed that PFOS suppressed D2 mRNA expression in the absence of T4 in primary cerebellar cells. D2 activity was also suppressed by PFOS in C6 glioma-derived cells. These results indicate that early-life exposure of PFOS disrupts TH-mediated cerebellar development possibly through the disruption of D2 activity and/or mRNA expression, which may cause cerebellar dysfunction.

TPO as an indicator of lymph node metastasis and recurrence in papillary thyroid carcinoma.

The objective of this study was to investigate the expression of thyroid peroxidase (TPO) in papillary thyroid carcinoma (PTC) and to preliminarily investigate its value as a marker of lymph node metastasis and recurrence in patients with PTC. Clinical data of PTC patients and TPO expression were collected from The Cancer Genome Atlas (TCGA) database for analysis. We recruited 230 consecutive PTC patients from the Department of Thyroid Surgery of the First Affiliated Hospital of Kunming Medical University, collected their clinicopathological data, and also performed immunohistochemical analysis of TPO expression on their thyroid specimens to validate the results of bioinformatics analysis. In addition, the construction of protein-protein interaction networks was performed too. Functional enrichment analysis and immuno-infiltration analysis characterized the pathways in which TPO genes may be involved. Data mining based on the TCGA database showed that TPO expression in PTC tissues was significantly lower than in paired normal thyroid tissues. The expression level of TPO in PTC tissues correlated with tumor lymph node metastasis and recurrence. Follow-up data from our center also validated the difference in TPO expression and its relationship with lymph node metastasis in PTC patients. Functional enrichment analysis showed that TPO function was significantly associated with signaling pathways related to amino acid metabolism, gene expression regulation and tumorigenesis. TPO expression was also significantly associated with immune infiltration. Our study showed that reduced TPO expression was significantly associated with lymph node metastasis and recurrence in patients with PTC, and we validated this result in our central cohort. These data suggest that TPO may serve as a prognostic indicator for PTC.

BDE209-promoted Dio2 degradation in H4 glioma cells through the autophagy pathway, resulting in hypothyroidism and leading to neurotoxicity.

Decabromodiphenyl ether (BDE209), the homologue with the highest number of brominates in polybrominated diphenyl ethers (PBDEs), is one of the most widespread environmental persistent organic pollutants (POPs) due to its mass production and extensive application in recent decades. BDE209 is neurotoxic, possibly related to its interference with the thyroid hormone (TH) system. However, the underlying molecular mechanisms of BDE209-induced TH interference and neurobehavioral disorders remains unknown. Here, we explored how BDE209 manipulated the major enzyme, human type II iodothyronine deiodinase (Dio2), that is most important in regulating local cerebral TH equilibrium by neuroglial cells, using an in vitro model of human glioma H4 cells. Clonogenic cell survival assay and LC/MS/MS analysis showed that BDE209 could induce chronic neurotoxicity by inducing TH interference. Co-IP assay, RT-qPCR and confocal assay identified that BDE209 destroyed the stability of Dio2 without affecting its expression, and promoted its binding to p62, thereby enhancing its autophagic degradation, thus causing TH metabolism disorder and neurotoxicity. Furthermore, molecular docking studies predicted that BDE209 could effectively suppress Dio2 activity by competing with tetraiodothyronine (T4). Collectively, our study demonstrates that BDE209-induced Dio2 degradation and loss of its enzymatic activity in neuroglial cells are the fundamental pathogenic basis for BDE209-mediated cerebral TH disequilibrium and neurotoxicity, providing a target of interest for further investigation using glial/neuronal cell co-culture system and in vivo models.

The Physiological Functions and Polymorphisms of Type II Deiodinase.

Type II deiodinase (DIO2) is thought to provide triiodothyronine (T3) to the nucleus to meet intracellular needs by deiodinating the prohormone thyroxine. DIO2 is expressed widely in many tissues and plays an important role in a variety of physiological processes, such as controlling T3 content in developing tissues (e.g., bone, muscles, and skin) and the adult brain, and regulating adaptive thermogenesis in brown adipose tissue (BAT). However, the identification and cloning of DIO2 have been challenging. In recent years, several clinical investigations have focused on the Thr92Ala polymorphism, which is closely correlated with clinical syndromes such as type 2 diabetes, obesity, hypertension, and osteoarthritis. Thr92Ala-DIO2 was also found to be related to bone and neurodegenerative diseases and tumors. However, relatively few reviews have synthesized research on individual deiodinases, especially DIO2, in the past 5 years. This review summarizes current knowledge regarding the physiological functions of DIO2 in thyroid hormone signaling and adaptive thermogenesis in BAT and the brain, as well as the associations between Thr92Ala-DIO2 and bone and neurodegenerative diseases and tumors. This discussion is expected to provide insights into the physiological functions of DIO2 and the clinical syndromes associated with Thr92Ala-DIO2.

Type 2 deiodinase is expressed in anaplastic thyroid carcinoma and its inhibition causes cell senescence.

Anaplastic thyroid cancer (ATC) is a rare thyroid tumor that frequently originates from the dedifferentiation of a well-differentiated papillary or follicular thyroid cancer. Type 2 deiodinase (D2), responsible for the activation of the thyroid hormone thyroxine into tri-iodothyronine (T3), is expressed in normal thyroid cells and its expression is strongly downregulated in papillary thyroid cancer. In skin cancer, D2 has been associated with cancer progression, dedifferentiation, and epithelial-mesenchymal transition. Here, we show that D2 is highly expressed in anaplastic compared to papillary thyroid cancer cell lines and that D2-derived T3 is required for ATC cell proliferation. D2 inhibition is associated with G1 growth arrest and induction of cell senescence, together with reduced cell migration and invasive potential. Finally, we found that mutated p5372R(R248W), frequently found in ATC, is able to induce D2 expression in transfected papillary thyroid cancer cells. Our results show that the action of D2 is crucial for ATC proliferation and invasiveness, providing a potential new therapeutic target for the treatment of ATC.

Longitudinal Associations Between TPO Gene Variants and Thyroid Peroxidase Antibody Seroconversion in a Population-Based Study: Tehran Thyroid Study.

Introduction: Autoimmune thyroid diseases (AITD) are usually accompanied by anti-thyroid antibodies which can serve as early predictive markers. This study was designed to investigate the relationship between thyroid peroxidase (TPO) gene variants and the presence of TPOAb and to evaluate the effect of environmental factors associated with seroconversion from TPOAb-negative to TPOAb-positive. Methods: Participants from phases 1 and 2 of the Tehran Thyroid Study in (n = 5327, ≥20 years) were evaluated in terms of TPOAb positivity, and its relationship with 53 single nucleotide polymorphisms (SNPs) from within the TPO gene (cross-sectional approach). TPOAb-negative participants (n = 4815) were followed up for seroconversion for 5.5 years. The relationship between the TPO gene variants and the TPOAb seroconversion was evaluated (longitudinal approach). Results: There were 521 TPOAb-positive participants in the cross-sectional phase and 266 new TPOAb-positive cases observed during the follow-up period. After quality control (Hardy-Weinberg equilibrium (p < 1 × 10-5) and minor allele frequency < 0.05), 49 SNPs were qualified for association analyses. From this set fourteen SNPs were identified that were associated with TPOAb positivity. rs6605278, located in the 3'UTR TPO gene, was the most highly significantly associated of the variant and remained associated after adjustment for age, gender, body mass index (BMI), smoking, number of parity, and oral contraceptive consumption in both cross-sectional and longitudinal analyses (p < 0.05). Conclusions: TPOAb-positivity can be partially explained by variants in the TPO gene. New TPOAb-associated SNPs were observed in Iranians as an ethnically diverse population.

Polymorphisms of Placental Iodothyronine Deiodinase Genes in a Rural Area of Northern China with High Prevalence of Neural Tube Defects.

INTRODUCTION: We have reported that high total homocysteine and the coexistence of inadequate thyroid hormones in maternal serum increase the risk of fetal neural tube defects (NTDs). Placental iodothyronine deiodinases (DIOs: DIO1, DIO2, and DIO3) play a role in regulating the conversions between different forms of maternal thyroid hormones. This study hypothesized that single nucleotide polymorphisms (SNPs) in placental DIOs genes could be related to NTDs. METHODS: We performed a case-control study from 2007 to 2009 that included pregnant women from Lüliang, Shanxi Province, China. Nine distinct SNPs in DIOs genes were analyzed, and placental samples were obtained from 83 pregnant women with NTD fetuses and 90 pregnant women with normal fetuses. The nine SNPs were analyzed using the Cochran-Armitage test and the Fisher's exact test. RESULTS: There were no statistically significant differences between case and control in the nine SNPs of DIOs (p > 0.05). CONCLUSIONS: The results of this study suggested that SNPs of DIO genes in the placenta among pregnant women have no statistically significant difference between the two groups, suggesting that other factors might be involved in metabolism of maternal thyroid hormone provided to fetuses, such as epigenetic modification of methylation and homocysteinylation and genomic imprinting in the placenta. Further functional studies on placenta samples are necessary.

Type 2 Deiodinase Thr92Ala Polymorphism and Aging Are Associated with a Decreased Pituitary Sensitivity to Thyroid Hormone.

Background: The DIO2 Thr92Ala polymorphism (rs225014), which occurs in about 15-30% of Caucasian people, determines a less efficient type 2 deiodinase (D2) enzyme. The aim of this study was to determine the impact of DIO2 Thr92Ala polymorphism on the serum thyrotropin (TSH) levels in thyroidectomized patients with hypothyroidism and to evaluate whether TSH levels and aging could be related, at pituitary level, to D2 activity. Methods: This prospective study was performed on 145 thyroid cancer patients, treated with total thyroidectomy, and undergoing radioiodine treatment after 3 weeks of levothyroxine (LT4) withdrawal. A mouse model has been used to determine D2 protein and mRNA levels in pituitary during aging. Results: Genetic analysis identified DIO2 Thr92Ala polymorphism in 56% of participants: 64/145 (44%) patients were homozygous wild type (WT) (Thr/Thr), 64 (44%) heterozygous (Thr/Ala), and 17 (12%) homozygous mutant (Ala/Ala). A significant negative relationship was observed between aging and the rise in serum TSH levels during LT4 withdrawal. However, this negative correlation found in WT was reduced in heterozygous and lost in mutant homozygous patients (Thr/Thr r = -0.45, p = 0.0002, 95% confidence interval [CI] -0.63 to -0.23; Ala/Thr r = -0.39, p = 0.0012, CI -0.60 to -0.67; and Ala/Ala r = -0.30, p = 0.2347; CI -0.70 to 0.20). Accordingly, when we compared the TSH measured in each patient to its theoretical value predicted from age, the TSH did not reach its putative target in 47% of WT patients, in 70% of Ala/Thr, and 76% of Ala/Ala carrying patients (p = 0.0036). This difference was lost in individuals older than 60 years, suggesting a decline of D2 associated with aging. The hypothesis that the pituitary D2 decreases with age was confirmed by the evidence that D2 mRNA and protein levels were lower in pituitary from old versus young mice. Conclusion: An age-related decline in TSH production in response to hypothyroidism was correlated with decreased D2 levels in pituitary. The presence of DIO2 homozygous Ala/Ala polymorphism was associated with a reduced level of TSH secretion in response to hypothyroidism, indicating a decreased pituitary sensitivity to serum thyroxine variation (Institutional Research Ethics board approval number no. 433/21).

Hypomethylation of Thyroid Peroxidase as a Biomarker for Hepatocellular Carcinoma with Tumor Thrombosis.

OBJECTIVE: Thyroid hormones (THs) regulate multiple physiological activities in the liver, including cellular metabolism, differentiation, and cell growth, and play important roles in the pathogenesis of hepatocellular carcinoma (HCC). Thyroid peroxidase (TPO) is a key molecule involved in the THs synthesis and signaling pathway. As an epigenetic modification, DNA methylation has a critical role in tumorigenesis with diagnostic potential. However, the connection between THs and DNA methylation has been rarely investigated. METHODS: The methylation of key TH-related genes was analyzed by in-house epigenome-wide scanning, and we further analyzed the methylation levels of the TPO promotor in 164 sample pairs of HCC and adjacent non-cancerous tissues by Sequenom EpiTYPER assays, and evaluated their clinical implications. RESULTS: We identified that the methylation of the TPO promoter was downregulated in the HCC tissues (P<0.0001) with a mean difference ranging from 18.5% to 22.3%. This methylation pattern correlated with several clinical factors, including a multi-satellite tumor, fibrous capsule, and the presence of tumor thrombus. The receiver operator characteristic (ROC) curve analysis further confirmed that the percent methylated reference (PMR) values for TPO were predictive of the tumor [the area under the curve (AUC) ranged from 0.755 to 0.818] and the thrombosis in the HCC patients (the AUC ranged from 0.706 to 0.777). CONCLUSION: These findings demonstrated that epigenetic alterations of TPO, as indicated by the PMR values, were a potential biomarker for HCC patients with tumor thrombosis.

Curating the gnomAD database: Report of novel variants in the thyroid peroxidase gene using in silico bioinformatics algorithms and a literature review.

Thyroid peroxidase (TPO) is a membrane-bound glycoprotein located at the apical side of the thyroid follicular cells that catalyzes both iodination and coupling of iodotyrosine residues within the thyroglobulin molecule, leading to the synthesis of thyroid hormone. Variants in TPO cause congenital hypothyroidism (CH) by iodide organification defect and are commonly inherited in an autosomal recessive fashion. In the present work, we report a detailed population analysis and bioinformatic prediction of the TPO variants indexed in the Genome Aggregation Database (gnomAD) v2.1.1. The proportion of missense cysteine variants and nonsense, frameshift, and splice acceptor/donor variants were analyzed in each ethnic group (European (Non-Finnish), European (Finnish), African/African Americans, Latino/Admixed American, East Asian, South Asian, Ashkenazi Jewish, Other). The results showed a clear predominance of frameshift variants in the East Asian (82%) and European (Finnish) (75%) population, whereas the splice site variants predominate in African/African Americans (99.46%), Other (96%), Latino/Admixed American (94%), South Asian (86%), European (Non-Finnish) (56%) and Ashkenazi Jewish (56%) populations. The analysis of the distribution of the variants indexed in gnomAD v2.1.1 database revealed that most missense variants identified in the An peroxidase domain map in exon 8, followed by exons 11, 7 and 9, and finally in descending order by exons 10, 6, 12 and 5. In total, 183 novel TPO variants were described (13 missense cysteine's variants, 158 missense variants involving the An peroxidase domain and 12 splicing acceptor or donor sites variants) which were not reported in the literature and that would have deleterious effects on prediction programs. In the gnomAD v2.1.1 population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:77. In conclusion, we provide an updated and curated reference source of new TPO variants for application in clinical diagnosis and genetic counseling. Also, this work contributes to elucidating the molecular basis of CH associated with TPO defects.

Type 2 deiodinase p.Thr92Ala polymorphism does not affect the severity of obesity and weight loss after bariatric surgery.

A single nucleotide polymorphism in the Type 2 deiodinase (DIO2) gene (p.Thr92Ala) was found to be associated with hypertension, type 2 diabetes mellitus (T2DM), insulin resistance, and body mass index (BMI). We retrospectively evaluated 182 patients to assess whether the DIO2 p.Thr92Ala was associated with severe obesity and response to bariatric surgery. Genomic DNA was extracted from peripheral blood leukocytes before surgery. Glycemic control parameters, cardiometabolic risk biomarkers (waist circumference, lipid assessment and blood pressure) and hormonal parameters were assessed at baseline and after surgery. Based on genotype evaluation, 78/182 (42.9%) patients were homozygous wild-type (Thr/Thr), 83/182 (45.6%) heterozygous (Thr/Ala), and 21/182 (11.5%) rare homozygous (Ala/Ala). Age at the time of the first evaluation in our Unit was significantly lower in patients with DIO2 p.Thr92Ala. No significant association was observed between DIO2 p.Thr92Ala and BMI, excess weight, waist circumference, Homa Index. The prevalence of comorbidities was not associated with allele distribution except for hypertension that was more frequent in wild-type patients (p = 0.03). After bariatric surgery, excess weight loss (EWL) % and remission from comorbidities occurred without differences according to genotypes. DIO2 p.Thr92Ala does not affect the severity of obesity and its complications, but it seems to determine an earlier onset of morbid obesity. The presence of polymorphism seems not to impact on the response to bariatric surgery, both in terms of weight loss and remission of comorbidities.

Selpercatinib-Induced Hypothyroidism Through Off-Target Inhibition of Type 2 Iodothyronine Deiodinase.

PURPOSE: The development of the selective RET inhibitors selpercatinib and pralsetinib has revolutionized the treatment of metastatic progressive RET-mutant medullary thyroid carcinoma (MTC) and other RET-driven cancers, given their more favorable side-effect profile. The aim of this study is to investigate the mechanisms of selpercatinib-induced thyroid dysfunction in athyreotic patients with RET-mutant MTC and in patients with RET-mutant non-small-cell lung cancer (NSCLC) who had a functional thyroid. MATERIALS AND METHODS: Thyroid hormone levels were evaluated in an observational cohort of five athyreotic patients with MTC and 30 patients with NSCLC before and after initiation of selpercatinib. In vitro experiments to identify the mechanism of selpercatinib-induced thyroid dysfunction were conducted in cells expressing endogenous D1, D2, and D3 iodothyronine deiodinases. RESULTS: Upon initiating treatment with selpercatinib, athyreotic patients developed clinical hypothyroidism with approximately 60% lower T3 levels despite adequate levothyroxine supplementation, whereas in patients with NSCLC, who retain a normal thyroid, selpercatinib resulted in a more attenuated reduction in serum T3, which was dose-dependent. We conducted studies in cells endogenously expressing either D1, D2, or D3, the three iodothyronine deiodinases. Selpercatinib inhibited D2-mediated T3 production in MSTO-211 cells by 50%. A modest repression of D2 mRNA was present in human thyroid cancer TT cells that express RET, but not in the MSTO-211 cells that do not. No effect of the drug was observed on D1 (activating deiodinase) or D3 (inactivating deiodinase). Thus, a nontranscriptional effect of selpercatinib on D2 activity is the most plausible explanation for the low T3 levels. CONCLUSION: An off-target effect of selpercatinib on D2-mediated T3 production leads to clinical hypothyroidism, primarily in levothyroxine-treated athyreotic patients. Liothyronine supplementation was needed to achieve normal T3 levels and restore clinical euthyroidism.

Autoantibodies against ATP4A are a feature of the abundant autoimmunity that develops in first-degree relatives of patients with type 1 diabetes.

OBJECTIVE: Type 1 diabetes is associated with autoantibodies to different organs that include the gut. The objective of the study was to determine the risk of developing gastric parietal cell autoimmunity in relation to other autoimmunity in individuals with a family history of type 1 diabetes. METHODS: Autoantibodies to the parietal cell autoantigen, H+ /K+ ATPase subunit A (ATP4A) was measured in 2218 first-degree relatives of patients with type 1 diabetes, who were prospectively followed from birth for a median of 14.5 years. All were also tested regularly for the development of islet autoantibodies, transglutaminase autoantibodies, and thyroid peroxidase autoantibodies. RESULTS: The cumulative risk to develop ATP4A autoantibodies was 8.1% (95% CI, 6.6-9.6) by age 20 years with a maximum incidence observed at age 2 years. Risk was increased in females (HR, 1.9; 95% CI, 1.3-2.8; p = 0.0004), relatives with the HLA DR4-DQ8/DR4-DQ8 genotype (HR, 3.4; 95% CI, 1.9-5.9; p < 0.0001) and in participants who also had thyroid peroxidase autoantibodies (HR, 3.7; 95% CI, 2.5-5.5; p < 0.0001). Risk for at least one of ATP4A-, islet-, transglutaminase-, or thyroid peroxidase-autoantibodies was 24.7% (95% CI, 22.6-26.7) by age 20 years and was 47.3% (95% CI, 41.3-53.3) in relatives who had an HLA DR3/DR4-DQ8, DR4-DQ8/DR4-DQ8, or DR3/DR3 genotype (p < 0.0001 vs. other genotypes). CONCLUSIONS: Relatives of patients with type 1 diabetes who have risk genotypes are at very high risk for the development of autoimmunity against gastric and other organs.

Insights into the Mechanism of Human Deiodinase 1.

The three isoenzymes of iodothyronine deiodinases (DIO1-3) are membrane-anchored homo-dimeric selenoproteins which share the thioredoxin-fold structure. Several questions regarding their catalytic mechanisms still remain open. Here, we addressed the roles of several cysteines which are conserved among deiodinase isoenzymes and asked whether they may contribute to dimerization and reduction of the oxidized enzyme with physiological reductants. We also asked whether amino acids previously identified in DIO3 play the same role in DIO1. Human DIO1 and 2 were recombinantly expressed in insect cells with selenocysteine replaced with cysteine (DIO1U126C) or in COS7 cells as selenoprotein. Enzyme activities were studied by radioactive deiodination assays with physiological reducing agents and recombinant proteins were characterized by mass spectrometry. Mutation of Cys124 in DIO1 prevented reduction by glutathione, while 20 mM dithiothreitol still regenerated the enzyme. Protein thiol reductants, thioredoxin and glutaredoxin, did not reduce DIO1U126C. Mass spectrometry demonstrated the formation of an intracellular disulfide between the side-chains of Cys124 and Cys(Sec)126. We conclude that the proximal Cys124 forms a selenenyl-sulfide with the catalytic Sec126 during catalysis, which is the substrate of the physiological reductant glutathione. Mutagenesis studies support the idea of a proton-relay pathway from solvent to substrate that is shared between DIO1 and DIO3.

Inactivation of Type 3 Deiodinase Results in Life-long Changes in the Brown Adipose Tissue Transcriptome in the Male Mouse.

Adaptive thermogenesis in small mammals and infants takes place in brown adipose tissue (BAT). Heat is produced via uncoupling protein 1 (UCP1)-mediated uncoupling between oxidation of energy substrates and adenosine 5'-triphosphate synthesis. Thyroid hormone (TH) signaling plays a role in this process. The deiodinases activate thyroxine (T4) to 3,5,3'-triiodothyronine (T3) (D2) or inactivate T4 and T3 to 3,3,5'-triiodothyronine and T2 (D3), respectively. Using a mouse model with selective inactivation of Dio3 in BAT (flox-Dio3 × UCP1-cre = BAT-D3KO), we now show that knocking out D3 resulted in premature exposure of developing brown adipocytes (embryonic days 16.5-18.5) to T3 signaling, leading to an earlier expression of key BAT genes, including Cidea, Cox8b, Dio2, Ucp1, and Pgc1α. Adult BAT-D3KO mice exhibited increased expression of 1591 genes as assessed by RNA sequencing, including 19 gene sets related to mitochondria, 8 related to fat, and 8 related to glucose homeostasis. The expression of 243 genes was changed by more than 1.5-fold, 36 of which play a role in metabolic/thermogenic processes. BAT-D3KO mice weigh less and exhibit smaller white adipocyte area, but maintain normal energy expenditure at room temperature (22 °C) and in the cold (4 °C). They also defend their core temperature more effectively and do not lose as much body weight when exposed to cold. We conclude that the coordinated actions of Dio3 in the embryonic BAT define the timing and intensity of T3 signaling during brown adipogenesis. Enhanced T3 signaling during BAT embryogenesis (Dio3 inactivation) results in selective life-long modifications in the BAT transcriptome.

Brown adipocyte ATF4 activation improves thermoregulation and systemic metabolism.

Cold-induced thermogenesis in endotherms demands adaptive thermogenesis fueled by mitochondrial respiration and Ucp1-mediated uncoupling in multilocular brown adipocytes (BAs). However, dietary regulation of thermogenesis in BAs isn't fully understood. Here, we describe that the deficiency of Leucine-rich pentatricopeptide repeat containing-protein (Lrpprc) in BAs reduces mtDNA-encoded ETC gene expression, causes ETC proteome imbalance, and abolishes the mitochondria-fueled thermogenesis. BA-specific Lrpprc knockout mice are cold resistant in a 4°C cold-tolerance test in the presence of food, which is accompanied by the activation of transcription factor 4 (ATF4) and proteome turnover in BAs. ATF4 activation genetically by BA-specific ATF4 overexpression or physiologically by a low-protein diet feeding can improve cold tolerance in wild-type and Ucp1 knockout mice. Furthermore, ATF4 activation in BAs improves systemic metabolism in obesogenic environment regardless of Ucp1's action. Therefore, our study reveals a diet-dependent but Ucp1-independent thermogenic mechanism in BAs that is relevant to systemic thermoregulation and energy homeostasis.