ASIC1a affects hypothalamic signaling and regulates the daily rhythm of body temperature in mice.

The body temperature of mice is higher at night than during the day. We show here that global deletion of acid-sensing ion channel 1a (ASIC1a) results in lower body temperature during a part of the night. ASICs are pH sensors that modulate neuronal activity. The deletion of ASIC1a decreased the voluntary activity at night of mice that had access to a running wheel but did not affect their spontaneous activity. Daily rhythms of thyrotropin-releasing hormone mRNA in the hypothalamus and of thyroid-stimulating hormone ß mRNA in the pituitary, and of prolactin mRNA in the hypothalamus and pituitary were suppressed in ASIC1a-/- mice. The serum thyroid hormone levels were however not significantly changed by ASIC1a deletion. Our findings indicate that ASIC1a regulates activity and signaling in the hypothalamus and pituitary. This likely leads to the observed changes in body temperature by affecting the metabolism or energy expenditure.

XB130 Deficiency Causes Congenital Hypothyroidism in Mice due to Disorganized Apical Membrane Structure and Function of Thyrocytes.

Background: Congenital hypothyroidism is often caused by genetic mutations that impair thyroid hormone (TH) production, resulting in growth and development defects. XB130 (actin filament associated protein 1 like 2) is an adaptor/scaffold protein that plays important roles in cell proliferation, migration, intracellular signal transduction, and tumorigenesis. It is highly expressed in thyrocytes, however, its function in the thyroid remains largely unexplored. Methods:Xb130-/- mice and their littermates were studied. Postnatal growth and growth hormone levels were measured, and responses to low or high-iodine diet, and levothyroxine treatment were examined. TH and thyrotropin in the serum and TH in the thyroid glands were quantified. Structure and function of thyrocytes in embryos and postnatal life were studied with histology, immunohistochemistry, immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction. Results:Xb130-/- mice exhibited transient growth retardation postnatally, due to congenital hypothyroidism with reduced TH synthesis and secretion, which could be rescued by exogenous thyroxine supplementation. The thyroid glands of Xb130-/- mice displayed diminished thyroglobulin iodination and release at both embryonic and early postnatal stages. XB130 was found mainly on the apical membrane of thyroid follicles. Thyroid glands of embryonic and postnatal Xb130-/- mice exhibited disorganized apical membrane structure, delayed folliculogenesis, and abnormal formation of thyroid follicle lumina. Conclusion: XB130 critically regulates folliculogenesis by maintaining apical membrane structure and function of thyrocytes, and its deficiency leads to congenital hypothyroidism.

Effects and molecular mechanism of pachymic acid on ferroptosis in renal ischemia reperfusion injury.

Acute kidney injury (AKI) is a common clinical disease. Ferropotosis, a new type of regulatory cell death, serves an important regulatory role in AKI. Pachymic acid (PA), a lanostane­type triterpenoid from Poria cocos, has been reported to be protective against AKI. However, the protective mechanism of PA in AKI is not yet fully understood. The present study aimed to investigate the effect and molecular mechanism of PA on ferroptosis in renal ischemia reperfusion injury in vivo. A total of 30 mice were intraperitoneally injected with 5, 10 and 20 mg/kg PA for 3 days. A bilateral renal pedicle clip was used for 40 min to induce renal ischemia­reperfusion injury and establish the model. The results demonstrated that treatment with PA decreased serum creatinine and blood urea nitrogen, and ameliorated renal pathological damage. Transmission electron microscopy revealed no characteristic changes in ferroptosis in the mitochondria of the renal tissue in the high­dose PA group, and only mild edema. Furthermore, treatment with PA increased glutathione expression, and decreased the expression levels of malondialdehyde and cyclooxygenase 2. Treatment with PA enhanced the protein and mRNA expression levels of the ferroptosis related proteins, glutathione peroxidase 4 (GPX4), solute carrier family 7 (cationic amino acid transporter, y+ system) member 11 (SLC7A11) and heme oxygenase 1 (HO­1) in the kidney, and increased the expression levels of nuclear factor erythroid derived 2 like 2 (NRF2) signaling pathway members. Taken together, the results of the present study suggest that PA has a protective effect on ischemia­reperfusion induced acute kidney injury in mice, which may be associated with the inhibition of ferroptosis in the kidneys through direct or indirect activation of NRF2, and upregulation of the expression of the downstream ferroptosis related proteins, GPX4, SLC7A11 and HO­1.

Homozygous loss-of-function mutations in SLC26A7 cause goitrous congenital hypothyroidism.

Defects in genes mediating thyroid hormone biosynthesis result in dyshormonogenic congenital hypothyroidism (CH). Here, we report homozygous truncating mutations in SLC26A7 in 6 unrelated families with goitrous CH and show that goitrous hypothyroidism also occurs in Slc26a7-null mice. In both species, the gene is expressed predominantly in the thyroid gland, and loss of function is associated with impaired availability of iodine for thyroid hormone synthesis, partially corrected in mice by iodine supplementation. SLC26A7 is a member of the same transporter family as SLC26A4 (pendrin), an anion exchanger with affinity for iodide and chloride (among others), whose gene mutations cause congenital deafness and dyshormonogenic goiter. However, in contrast to pendrin, SLC26A7 does not mediate cellular iodide efflux and hearing in affected individuals is normal. We delineate a hitherto unrecognized role for SLC26A7 in thyroid hormone biosynthesis, for which the mechanism remains unclear.

TRH Action Is Impaired in Pituitaries of Male IGSF1-Deficient Mice.

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function expressed in thyrotropin (TSH)-producing thyrotrope cells of the anterior pituitary gland. The protein is cotranslationally cleaved, with only its C-terminal domain (CTD) being trafficked to the plasma membrane. Most intragenic IGSF1 mutations in humans map to the CTD. In this study, we used CRISPR-Cas9 to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele encodes a truncated protein that fails to traffic to the plasma membrane. Under standard laboratory conditions, Igsf1-deficient males exhibit normal serum TSH levels as well as normal numbers of TSH-expressing thyrotropes. However, pituitary expression of the TSH subunit genes and TSH protein content are reduced, as is expression of the receptor for thyrotropin-releasing hormone (TRH). When challenged with exogenous TRH, Igsf1-deficient males release TSH, but to a significantly lesser extent than do their wild-type littermates. The mice show similarly attenuated TSH secretion when rendered profoundly hypothyroid with a low iodine diet supplemented with propylthiouracil. Collectively, these results indicate that impairments in pituitary TRH receptor expression and/or downstream signaling underlie central hypothyroidism in IGSF1 deficiency syndrome.

The syndrome of inherited partial SBP2 deficiency in humans.

Selenium (Se) is an essential trace element required for the biosynthesis of selenoproteins. Selenocysteine insertion sequence (SECIS) binding protein 2 (SBP2) represents a key trans-acting factor for the co-translational insertion of selenocysteine into selenoproteins. In 2005, we reported the first mutations in the SBP2 gene in two families in which the probands presented with transient growth retardation associated with abnormal thyroid function tests. Intracellular metabolism of thyroid hormone (TH) and availability of the active hormone, triiodothyronine, is regulated by three selenoprotein iodothyronine deiodinases (Ds). While acquired changes in D activities are common, inherited defects in humans were not known. Affected children were either homozygous or compound heterozygous for SBP2 mutations. Other selenoproteins, glutathione peroxidase, and selenoprotein P were also reduced in affected subjects. Since our initial report, another family manifesting a similar phenotype was found to harbor a novel SBP2 mutation. In vivo studies of these subjects have explored the effects of Se and TH supplementation. In vitro experiments have provided new insights into the effect of SBP2 mutations. In this review we discuss the clinical presentation of SBP2 mutations, their effect on protein function, consequence for selenoproteins, and the clinical course of subjects with SBP2 defects.

Clinical and molecular characterization of a novel selenocysteine insertion sequence-binding protein 2 (SBP2) gene mutation (R128X).

CONTEXT: Although acquired abnormalities of thyroid hormone metabolism are common, inherited defects in humans involving the synthesis of selenoproteins, including iodothyronine deiodinases, have been described in only one recent publication. OBJECTIVE: We report the study of a novel selenocysteine insertion sequence-binding protein 2 (SBP2) gene mutation (R128X) and its clinical and molecular characterization. SUBJECTS AND METHODS: A family of African origin was studied. The proband presented with growth retardation, low serum selenium level, and thyroid test abnormalities consisting of high serum total and free T(4) concentrations associated with low T(3), high rT(3), and normal TSH. The entire coding region of the SBP2 gene was sequenced and minigenes constructed to explain the nature of the defect. RESULTS: The proband was homozygous for a nonsense gene mutation that produces an early stop codon (R128X). Both parents and a sister were heterozygous but showed no growth or thyroid test abnormalities. Despite the severity of the defect, the patient had a relatively mild phenotype, similar to that associated with partial SBP2 deficiency. In vitro analysis showed that the mutant minigene synthesized SBP2 from at least three downstream ATGs capable of generating molecules containing the essential functional domains. Treatment with l-T(3) accelerated the growth velocity and advanced the bone age. CONCLUSIONS: We identified a novel SBP2 gene mutation producing an early arrest in the synthesis of a full-length molecule. The demonstration that SBP2 isoforms containing all functional domains could be synthesized from three downstream ATGs explains the relatively mild phenotype caused by this defect.

Selenium supplementation fails to correct the selenoprotein synthesis defect in subjects with SBP2 gene mutations.

BACKGROUND: Selenium (Se) is an essential trace element needed for the biosynthesis of selenoproteins. Selenocysteine incorporation sequence binding protein 2 (SBP2) represents a key trans-acting factor for the co-translational insertion of selenocysteine into selenoproteins. We recently described children with mutations in the SBP2 gene who displayed abnormal thyroid function tests and reduced selenoprotein concentrations. We have tried to improve selenoprotein biosynthesis and thyroid hormone metabolism in SBP2 deficient subjects by supplementing an organic and an inorganic Se form. METHODS: Three affected and two unaffected siblings received daily doses of 100, 200, or 400 microg selenomethionine-rich yeast and 400 microg sodium selenite for one month each. Serum was drawn at baseline and after supplementations. Thyroid function tests, extracellular glutathione peroxidase activity, Se, and selenoprotein P concentrations were determined. RESULTS: Selenomethionine-rich yeast increased serum Se concentrations in all subjects irrespective of genotype. Sodium selenite was effective in increasing the selenoprotein P concentration in normal and to a lesser degree in affected subjects. Both forms failed to increase the glutathione peroxidase activity or to correct the thyroid function abnormalities in the SBP2 deficient individuals indicating that impaired deiodinase expression was not positively affected. No adverse side effects were observed. CONCLUSIONS: Total serum Se concentrations in SBP2 deficient subjects respond to selenomethionine supplementation but this effect is not indicative for improved selenoprotein synthesis. Se is obviously not a limiting factor in the SBP2 deficient individuals when regular daily Se intake is provided. These findings might help to identify and diagnose more individuals with selenoprotein biosynthesis defects who might present at young age irrespective of their Se supply with characteristic thyroid function test abnormalities, growth retardation, and reduced Se and selenoprotein concentrations.

Type 3 deiodinase deficiency results in functional abnormalities at multiple levels of the thyroid axis.

The type 3 deiodinase (D3) is a selenoenzyme that inactivates thyroid hormones and is highly expressed during development and in the adult central nervous system. We have recently observed that mice lacking D3 activity (D3KO mice) develop perinatal thyrotoxicosis followed in adulthood by a pattern of hormonal levels that is suggestive of central hypothyroidism. In this report we describe the results of additional studies designed to investigate the regulation of the thyroid axis in this unique animal model. Our results demonstrate that the thyroid and pituitary glands of D3KO mice do not respond appropriately to TSH and TRH stimulation, respectively. Furthermore, after induction of severe hypothyroidism by antithyroid treatment, the rise in serum TSH in D3KO mice is only 15% of that observed in wild-type mice. In addition, D3KO animals rendered severely hypothyroid fail to show the expected increase in prepro-TRH mRNA in the paraventricular nucleus of the hypothalamus. Finally, treatment with T(3) results in a serum T(3) level in D3KO mice that is much higher than that in wild-type mice. This is accompanied by significant weight loss and lethality in mutant animals. In conclusion, the absence of D3 activity results in impaired clearance of T(3) and significant defects in the mechanisms regulating the thyroid axis at all levels: hypothalamus, pituitary, and thyroid.

Tissue-specific thyroid hormone deprivation and excess in monocarboxylate transporter (mct) 8-deficient mice.

Mutations of the X-linked thyroid hormone (TH) transporter (monocarboxylate transporter, MCT8) produce in humans unusual abnormalities of thyroid function characterized by high serum T3 and low T4 and rT3. The mechanism of these changes remains obscure and raises questions regarding the regulation of intracellular availability and metabolism of TH. To study the pathophysiology of MCT8 deficiency, we generated Mct8 knockout mice. Male mice deficient in Mct8 (Mct8(-/y)) replicate the thyroid abnormalities observed in affected men. TH deprivation and replacement with L-T3 showed that suppression of TSH required higher serum levels T3 in Mct8(-/y) than wild-type (WT) littermates, indicating hypothalamus and/or thyrotroph resistance to T3. Furthermore, T4 is required to maintain the high serum T3 level because the latter was not different between the two genotypes during administration of T3. Mct8(-/y) mice have 2.3-fold higher T3 content in liver associated with 6.1- and 3.1-fold increase in deiodinase 1 mRNA and enzymatic activity, respectively. The relative T3 excess in liver of Mct8(-/y) mice produced a decrease in serum cholesterol (79 +/- 18 vs. 137 +/- 38 mg/dl in WT) and an increase in alkaline phosphatase (107 +/- 23 vs. 58 +/- 3 U/liter in WT) levels. In contrast, T3 content in cerebrum was 1.8-fold lower in Mct8(-/y) mice, associated with a 1.6- and 10.6-fold increase in D2 mRNA and enzymatic activity, respectively, as previously observed in TH-deprived WT mice. We conclude that cell-specific differences in intracellular TH content due to differences in contribution of the various TH transporters are responsible for the unusual clinical presentation of this defect, in contrast to TH deficiency.

Dominant role of thyrotropin-releasing hormone in the hypothalamic-pituitary-thyroid axis.

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the beta isoforms of TH receptors (TRbeta KO), or both (double KO). TRbeta knock-out (KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.

Hypothyroidism in thyroid transcription factor 1 haploinsufficiency is caused by reduced expression of the thyroid-stimulating hormone receptor.

Humans expressing one allele of the thyroid transcription factor 1 (TTF1) gene have neurological symptoms and increased serum TSH with variable degrees of hypothyroidism. Ttf1+/- mice have also poor coordination and increased serum TSH concentration (205 +/- 22 vs. 92 +/- 12 mU/liter; P < 0.001) and slightly lower T4 (46 +/- 3 vs. 63 +/- 6 nmol/liter; P < 0.02) as compared with Ttf1+/+ mice. To determine whether the hypothyroidism is of central or primary origin, we examined the bioactivity of TSH, thyroidal response to exogenous TSH and the expression of genes regulated by TTF1. TSH bioactivity was normal, but T4 response to a low but not high dose of TSH was significantly reduced in the Ttf1+/- mice (5.5 +/- 2.2 vs. 15.3 +/- 4.1 nmol/liter; P < 0.03), indicating a reduced thyroidal response. Thyroid mRNAs were measured by real-time PCR (Ttf1+/+ littermates = 100%). Ttf1+/- mice had half the levels of TTF1 mRNA (54 +/- 9; P < 0.01) and protein, confirming their haploinsufficiency. Significantly lower levels of mRNAs were observed for two of the three genes with TTF1 cis elements: TSH receptor (TSHr, 57 +/- 4%; P < 0.002), thyroglobulin (63 +/- 7%; P < 0.005), but not thyroid peroxidase (81 +/- 12%; P > 0.05). No significant difference between the two genotypes was found for Pax8, sodium iodide symporter, and iodothyronine deiodinase 1. These results show that Ttf1 haploinsufficiency causes a reduction in the expression of TSHr and thyroglobulin, genes with TTF1 binding sites in their promoter regions. The low TSHr is only partially compensated by an increase in TSH secretion because T4 remains mildly reduced. However, administration of a larger amount of TSH obliterates the response differences by saturating a reduced amount of receptor.