ABSENCE OF A THYROID PHENOTYPE IN SORTILIN-DEFICIENT MICE.

OBJECTIVE: The Vps10p family member sortilin is expressed in thyroid epithelial cells where it contributes to recycling of the thyroid hormone precursor thyroglobulin (Tg), a process that is thought to render hormone release more effective. Here we investigated the functional impact of sortilin in the thyroid gland using sortilin-deficient mice. METHODS: We measured free T4, thyroid-stimulating hormone (TSH) and Tg serum levels and studied thyroid morphology in 14 sortilin-deficient (Sort1)(-/-)and 12 wildtype (WT) mice. RESULTS: Serum free T4 levels did not differ between Sort1(-/-)and WT females but were significantly lower in Sort1(-/-)males compared with WT (P = .0424). Neither serum TSH nor Tg levels differed between Sort1(-/-)and WT mice, regardless of sex. On the same line, no thyroid histology differences were observed. CONCLUSION: Our findings seem to exclude a role of sortilin in thyroid hormone secretion, although it is possible that the absence of sortilin may result in a thyroid phenotype if combined with other molecular defects of thyroid hormone synthesis and secretion or under iodine deficiency.

Thyroidal effect of metformin treatment in patients with polycystic ovary syndrome.

OBJECTIVE: Metformin is widely used for the treatment of type 2 diabetes. Growing evidence supports the beneficial effects of metformin also in patients with polycystic ovary syndrome (PCOS). It was recently reported that metformin has a TSH-lowering effect in hypothyroid patients with diabetes being treated with metformin. DESIGN: Aim of this study was to evaluate the effect of metformin treatment on the thyroid hormone profile in patients with PCOS. PATIENTS AND MEASUREMENTS: Thirty-three patients with PCOS were specifically selected for being either treated with levothyroxine for a previous diagnosis of hypothyroidism (n = 7), untreated subclinically hypothyroid (n = 2) or euthyroid without levothyroxine treatment (n = 24) before the starting of metformin. The serum levels of TSH and FT(4) were measured before and after a 4-month period of metformin therapy. RESULTS: Thyroid function parameters did not change after starting metformin therapy in euthyroid patients with PCOS. In the 9 hypothyroid patients with PCOS, the basal median serum levels of TSH (3·2 mIU/l, range = 0·4-7·1 mIU/l) significantly (P < 0·05) decreased after a 4-month course of metformin treatment (1·7 mIU/l, range = 0·5-5·2 mIU/l). No significant change in the serum levels of FT4 was observed in these patients. The TSH-lowering effect of metformin was not related to the administered dose of the drug, which was similar in euthyroid as compared with hypothyroid patients with PCOS (1406 ± 589 vs 1322 ± 402 mg/day, respectively; NS). CONCLUSIONS: These results indicate that metformin treatment has a TSH-lowering effect in hypothyroid patients with PCOS, both treated with l-thyroxine and untreated.

Sortilin is a putative postendocytic receptor of thyroglobulin.

The Vps10p family member sortilin is involved in various cell processes, including protein trafficking. Here we found that sortilin is expressed in thyroid epithelial cells (thyrocytes) in a TSH-dependent manner, that the hormone precursor thyroglobulin (Tg) is a high-affinity sortilin ligand, and that binding to sortilin occurs after Tg endocytosis, resulting in Tg recycling. Sortilin was found to be expressed intracellularly in thyrocytes, as observed in mouse, human, and rat thyroid as well as in FRTL-5 cells. Sortilin expression was demonstrated to be TSH dependent, both in FRTL-5 cells and in mice treated with methimazole and perchlorate. Plasmon resonance binding assays showed that Tg binds to sortilin in a concentration-dependent manner and with high affinity, with Kd values that paralleled the hormone content of Tg. In addition, we found that Tg and sortilin interact in vivo and in cultured cells, as observed by immunoprecipitation, in mouse thyroid extracts and in COS-7 cells transiently cotransfected with sortilin and Tg. After incubation of FRTL-5 cells with exogenous, labeled Tg, sortilin and Tg interacted intracellularly, presumably within the endocytic pathway, as observed by immunofluorescence and immunoelectron microscopy, the latter technique showing some degree of Tg recycling. This was confirmed in FRTL-5 cells in which Tg recycling was reduced by silencing of the sortilin gene and in CHO cells transfected with sortilin in which recycling was increased. Our findings provide a novel pathway of Tg trafficking and a novel function of sortilin in the thyroid gland, the functional impact of which remains to be established.

TSH-Dependent expression of the LDL receptor-associated protein (RAP) in thyroid epithelial cells.

The low density lipoprotein (LDL) receptor-associated protein (RAP) is an endoplasmic reticulum (ER)-resident molecular chaperone for several LDL receptor family members and it also binds to thyroglobulin (Tg), the thyroid hormone precursor. Disruption of the RAP gene in thyrocytes results in impaired Tg secretion. To gain further insights into the function of RAP in the thyroid, we investigated whether its expression in thyrocytes is regulated by thyroid-stimulating hormone (TSH), a feature common to all proteins involved in thyroid hormone secretion. We found by immunofluorescence that in FRTL-5 cells cultured in the presence of TSH, RAP is expressed intracellularly. The levels of expression increased after exposure to TSH, beginning at 48 hours, in a concentration-dependent manner as observed by immunofluorescence and Western blotting. Expression of RAP was also increased by TSH in primary cultures of human thyrocytes as observed by Western blotting. In hypothyroid mice with high serum TSH, RAP was markedly increased compared with euthyroid mice as observed by immunohistochemistry and Western blotting. Based on these findings, we concluded that RAP is expressed by thyrocytes in a TSH-dependent manner, both in cultured thyroid cells and in vivo.

Thyroid dysfunction in megalin deficient mice.

Megalin mediates transcytosis of thyroglobulin (Tg), the thyroid hormone precursor, resulting in its passage into the bloodstream. The process involves especially hormone-poor Tg, which may favour hormone secretion by preventing competition with hormone-rich Tg for proteolytic degradation. To gain more insight into the role of megalin, here we studied thyroid function and histology in megalin deficient mice compared with WT mice. As expected from the knowledge that megalin mediates Tg transcytosis, serum Tg levels were significantly reduced in homozygous (megalin-/-) mice, which, more importantly, were found to be hypothyroid, as demonstrated by significantly reduced serum free thyroxine and significantly increased serum thyroid stimulating hormone (TSH) levels. In heterozygous (megalin+/-) mice, in which megalin expression was normal, thyroid function was unaffected. Although the serological phenotype in megalin-/- mice was not associated with histological alterations or goiter, our results support a major role of megalin in thyroid hormone secretion.

Enalapril reduces proliferation and hyaluronic acid release in orbital fibroblasts.

BACKGROUND: Orbital fibroblast proliferation and hyaluronic acid (HA) release are responsible for some of the clinical features of Graves' ophthalmopathy (GO). Thus, inhibition of these processes may be a possible therapeutic approach to this syndrome. Enalapril, a widely used antihypertensive drug, was found to have some inhibitory actions on fibroblast proliferation in cheloid scars in vivo, based on which we investigated its effects in primary cultures of orbital fibroblasts from GO patients and control subjects. METHODS: Primary cultures of GO and control fibroblasts were treated with enalapril or with a control compound (lisinopril). Cell proliferation assays, lactate dehydrogenase release assays (as a measure of cell necrosis), apoptosis assays, and measurement of HA in the cell media were performed. RESULTS: Enalapril significantly reduced cell proliferation in both GO and control fibroblasts. Because enalapril did not affect cell necrosis and apoptosis, we concluded that its effects on proliferation reflected an inhibition of cell growth and/or a delay in cell cycle. Enalapril significantly reduced HA concentrations in the media from both GO and control fibroblasts. CONCLUSIONS: Enalapril has antiproliferative and HA suppressing actions in both GO and control fibroblasts. Clinical studies are needed to investigate whether enalapril has any effects in vivo in patients with GO.

ClC-5 does not affect megalin expression and function in the thyroid.

Megalin is an endocytic receptor responsible for thyroglobulin (Tg) transcytosis, a process that favors hormone release. Accordingly, megalin KO mice have primary hypothyroidism. In the kidney, megalin expression is reduced when the gene encoding the chloride transporter ClC-5 is mutated. We investigated whether megalin expression and function in the thyroid are affected by ClC-5 using a ClC-5 KO mouse model. By Western blotting, ClC-5 was found in thyroid tissue extracts of WT, but not of ClC-5 KO mice. In addition, ClC-5 was found to be expressed by cultured thyroid cells (FRTL-5). The thyroid size, weight, and histology were similar in ClC- 5 KO and WT mice, as were the amounts of megalin in thyroid extracts. Accordingly, serum Tg, a measure of megalin-mediated transcytosis, was similar in WT and ClC-5 KO mice, suggesting that megalin function was unaffected. Thus, unlike in megalin KO mice, in ClC-5 KO mice thyroid function was unchanged, as indicated by the normal serum FT4 and TSH. We concluded that in the thyroid, unlike in the kidney, ClC-5 does not affect megalin expression and function, suggesting that megalin is differentially regulated in these two organs.

Defective thyroglobulin storage in LDL receptor-associated protein-deficient mice.

The molecular chaperone receptor-associated protein (RAP) is required for biosynthesis of megalin, an endocytic receptor for follicular thyroglobulin (Tg), the thyroid hormone precursor. RAP also binds to Tg itself, suggesting that it may affect Tg trafficking in various manners. To elucidate RAP function, we have studied the thyroid phenotype in RAP-knockout (RAP-KO) mice and found a reduction of Tg aggregates into thyroid follicles. Serum Tg levels were significantly increased compared with those of wild-type (WT) mice, suggesting a directional alteration of Tg secretion. In spite of these abnormalities, hormone secretion was maintained as indicated by normal serum thyroxine levels. Because Tg in thyroid extracts from RAP-KO mice contained thyroxine residues as in WT mice, we concluded that in RAP-KO mice, follicular Tg, although reduced, was nevertheless sufficient to provide normal hormone secretion. Serum TSH was increased in RAP-KO mice, and although no thyroid enlargement was observed, some histological features resembling early goiter were present. Megalin was decreased in RAP-KO mice, but this did not affect thyroid function, probably because of the concomitant reduction of follicular Tg. In conclusion, RAP is required for the establishment of Tg reservoirs, but its absence does not affect hormone secretion.