The deterioration of glucose metabolism frequently observed in hyperthyroidism may be due in part to increased gluconeogenesis in the liver and glucose efflux through hepatocyte plasma membranes. Glucose transporter 2 (GLUT 2), a facilitative glucose transporter localized to the liver and pancreas, may play a role in this distorted glucose metabolism. We examined changes in the levels of GLUT 2 in livers from rats with l-thyroxine-induced hyperthyroidism or methimazole-induced hypothyroidism by using Western blotting to detect GLUT 2. An oral glucose tolerance test revealed an oxyhyperglycemic curve (impaired glucose tolerance) in hyperthyroid rats (n=7) and a flattened curve in hypothyroid rats (n=7). GLUT 2 levels in hepatocyte plasma membranes were significantly increased in hyperthyroid rats and were not decreased in hypothyroid rats compared with euthyroid rats. The same results were obtained with a densitometric assay. These findings suggest that changes in the liver GLUT 2 concentration may contribute to abnormal glucose metabolism in thyroid disorders.
T Mokuno, K Uchimura, R Hayashi, N Hayakawa, M Makino, M Nagata, H Kakizawa, Y Sawai, M Kotake, N Oda, A Nakai, A Nagasaka and M Itoh
N. Hoogerbrugge–v.d.Linden, H. Jansen, W. C. Hülsmann and J. C. Birkenhäger
The effect of insulin-like growth factor-I (IGF-I) on the disturbance of lipid metabolism during primary hypothyroidism was studied in 12 women with primary hypothyroidism. Significant increases in both low-density lipoprotein (LDL) cholesterol and intermediate-density lipoprotein cholesterol were seen. Lipoprotein concentrations reverted to normal after substitution with thyroxine (T4) until the euthyroid state was reached. A decrease in IGF-I of 65% (P<0·005) was seen in hypothyroid patients and this was inversely correlated (r=−0·75; P<0·01) with the concentration of LDL cholesterol. Multivariate regression analysis of LDL cholesterol against IGF-I and free T4 showed that IGF-I determines the concentration of LDL cholesterol instead of free T4.
Our data suggest that in hypothyroidism, IGF-I is a determinant of the concentration of LDL cholesterol. In addition, hypothyroidism can influence plasma lipoprotein metabolism by lowering the activity of the salt-resistant lipase (liver lipase).
Journal of Endocrinology (1989) 123, 341–345
Beate Karges, Gerd Krause, Janos Homoki, Klaus-Michael Debatin, Nicolas de Roux and Wolfram Karges
Mutations of the human thyrotrophin receptor (TSH-R) are a cause of thyroid adenomas and hyperthyroidism. Here we study mechanisms of receptor activation in a genomic TSH-R variant V509A located in transmembrane helix (TMH) 3, which we identify in a family with congenital hyperthyroidism, multiple adenomas and follicular thyroid cancer. Using molecular modelling and dynamic simulation, we predicted the release of amino acid residue A593 (located opposite in domain TMH5) from a tight ‘knob-and-hole’ interaction with TMH3, physiologically constrained in the native receptor state by the bulky side chain of V509. To experimentally validate this concept, we generated mutant TSH-R expression constructs for functional in vitro studies. TSH-R mutant V509A showed a 2.8-fold increase in basal cAMP production, confirming constitutive TSH-R activation. The addition of a second site suppressor mutant A593V to TSH-R V509A resulted in the normalization of basal cAMP release, and the dose-responsiveness to TSH ligand was maintained. These data thus demonstrate that TSH-R V509A activation is caused by the release of TMH3–TMH5 interhelical constraints, while the native TSH-R conformation is re-stabilized by the introduction of a spacious valine residue at position 593. In conclusion, we delineate a novel mechanism of constitutive TSH-R activation, leading to thyroid hyperfunction and neoplasia.
Takao Ando, Rauf Latif and Terry F Davies
The post-translational processing of the TSH receptor (TSHR) includes intra-molecular cleavage with the loss of a 50 amino acid ectodomain region and the formation of two subunits (α and β), followed by likely α subunit shedding. TSHR antibodies (TSHR-Abs), which are directed at the ectodomain, may influence thyroid function by stimulating or inhibiting TSHR signaling or may bind without any such influence (the neutral group of antibodies). When we examined the characteristics of a series of monoclonal TSHR-Abs, we found that many were able to inhibit receptor cleavage and enhance TSHR expression. This was especially apparent with the neutral type of TSHR-Abs directed to the cleaved region of the ectodomain (aa 316–366). Indeed, such inhibition appeared to be epitope dependent with TSHR-Abs directed to regions after residues 335–354 showing no such activity. We propose that this aberrant process, whereby TSHR-Abs influence antigen processing, is a novel mechanism for the maintenance and exacerbation of autoimmune thyroid disease.
M Theodoropoulou, T Arzberger, Y Gruebler, Z Korali, P Mortini, W Joba, AE Heufelder, GK Stalla and L Schaaf
Thyrotrophin (TSH) synthesis and secretion is under the positive control of thyrotrophin releasing hormone and under the negative control of the thyroid hormones. However, it is hypothesised that TSH has a direct effect on the regulation of its own synthesis through an intrapituitary loop mediated by pituitary TSH receptors (TSH-R). The aim of this investigation was to study the expression of TSH-R in normal human pituitary at mRNA and protein levels, and to compare the pattern of protein expression between different pituitary adenomas. Using RT-PCR we were able to detect TSH-R mRNA in the normal pituitary, and immunohistochemical studies showed TSH-R protein expression in distinct areas of the anterior pituitary. Double immunostaining with antibodies against each of the intrapituitary hormones and S100 revealed that TSH-R protein is present in thyrotrophs and folliculostellate cells. Examination of 58 pituitary adenomas, including two clinically active and two clinically inactive thyrotroph adenomas, revealed TSH-R immunopositivity in only the two clinically inactive thyrotroph adenomas. This study shows, for the first time, the presence of TSH-R protein in the normal anterior pituitary and in a subset of thyrotroph adenomas. The expression of TSH-R in the thyrotroph and folliculostellate cell subpopulations provides preliminary evidence of a role for TSH in autocrine and paracrine regulatory pathways within the anterior pituitary gland.
T Mano, K Iwase, Y Sawai, N Oda, Y Nishida, T Mokuno, Y Itoh, M Kotake, R Masunaga, A Nakai, T Tujimura, A Nagasaka and H Hidaka
To investigate the effect of thyroid hormone on cardiac muscle dysfunction in hyper- and hypothyroid states, we evaluated cyclic 3′, 5′-nucleotide metabolism by measuring cyclic 3′, 5′-nucleotide phosphodiesterase activity and calmodulin concentrations in the cardiac muscles of hyper- and hypothyroid rats.
Cyclic AMP (cAMP) concentration was significantly high in the cardiac muscle of hyperthyroid rats and low in that from hypothyroid rats compared with control rats. Cyclic AMP and cyclic GMP phosphodiesterase activities were significantly decreased in the soluble fraction of cardiac muscle from hyperthyroid rats and markedly increased in this fraction in hypothyroid rats compared with normal animals. Calmodulin concentration was high in hyperthyroid and low in hypothyroid rats.
It was concluded from these findings that low cAMP-phosphodiesterase activity might, in part, bring about the high concentration of cAMP. Calmodulin was sigificantly high in the cardiac muscle of hyperthyroid rats and the reverse was the case in hypothyroid rats compared with normal rats. The implication is that, in hyper- and hypothyroid states, these changes may play an important role in cardiac function via their effect on cyclic nucleotide and Ca2+ metabolism.
Journal of Endocrinology (1994) 143, 515–520
Shuang-Xia Zhao, Shanli Tsui, Anthony Cheung, Raymond S Douglas, Terry J Smith and J Paul Banga
The TSH receptor (TSHR) is the critical target for antibody production in Graves' disease (GD). Insulin-like growth factor 1 receptor (IGF1R) has been proposed as a second autoantigen in complications of GD such as orbitopathy. We attempted to induce orbital tissue remodeling in mice undergoing immunizations with plasmids encoding TSHR and IGF1R delivered by in vivo skeletal muscle electroporation, a procedure known to give a sustained, long-term antibody response. Female BALB/c mice were challenged with TSHR A-subunit or IGF1Rα subunit plasmid by injection and electroporation. Mice challenged with TSHR A-subunit plasmid resulted in high frequency (75%) of hyperthyroidism and thyroid-stimulating antibodies. But strikingly, immunization with TSHR A-subunit plasmid also elicited antibody to IGF1Rα subunit. Mice challenged in the same manner with IGF1Rα subunit plasmid produced strong antibody responses to IGF1R, but did not undergo any changes in phenotype. Simultaneous challenge by double antigen immunization with the two plasmids in distant anatomical sites reduced the incidence of hyperthyroidism, potentially as a consequence of antigenic competition. Thyroid glands from the TSHR A-subunit plasmid-challenged group were enlarged with patchy microscopic infiltrates. Histological analysis of the orbital tissues demonstrated moderate connective tissue fibrosis and deposition of Masson's trichrome staining material. Our findings imply that immunization with TSHR A-subunit plasmid leads to generation of IGF1R antibodies, which together with thyroid-stimulating antibodies may precipitate remodeling of orbital tissue, raising our understanding of its close association with GD.
M L Panno, E Beraldi, V Pezzi, M Salerno, G De Luca, M Lanzino, M Le Pera, D Sisci, M Prati, S Palmero, E Bolla, E Fugassa and S Andò
The aim of the present study was to investigate the influence of thyroid hormones on androgen metabolism in Sertoli cells isolated from 3- and 4- week-old rats.
Hypothyroidism was induced by the oral administration of 0·025% methimazole (MMI) from birth until the rats were killed at 3 and 4 weeks of age. Half of the MMI-treated animals were injected i.p. with l-tri-iodothyronine (T3 3 μg/100 g body weight) during the last week before death. Sertoli cells from all groups were initially cultured under basal conditions for the first 24 h and subsequently in the presence of testosterone with or without T3 for an additional 24 h. Hypothyroidism was associated with severe impairment of body as well as testicular growth. Indeed, body and testicular weights were similar in 4-week-old hypothyroid animals to those in 3-week-old control rats.
Testosterone metabolism in Sertoli cells isolated from 3- and 4-week-old hypothyroid rats was mainly expressed by the lowering of 5α-dihydrotestosterone + androstane 3α, 17β–diol and an enhanced formation of 5α-reduced steroids with poor androgenic properties (e.g. 5α–androstane, 3, 17α-dione (androstanedione), 5α–androstan, 3-ol-17-one (androsterone)). Treatment of the same group of animals with T3 in vivo and in vitro did not influence the pattern of 5α–reductase steroids substantially.
The most striking finding in the Sertoli cells of 3-week-old hypothyroid rats was the dramatic enhancement of oestradiol formation which persisted to a lesser extent 1 week later. Oestradiol formation was greatly decreased by the addition of T3 in vivo and in vitro in hypothyroid animals.
These results suggest that T3 might influence androgen metabolism during the functional maturation of Sertoli cells.
Journal of Endocrinology (1994) 140, 349–355
ST Chen, JD Lin and KH Lin
The expression of TSH receptor (TSHR) gene is frequently lost in thyroid cancers during the process of dedifferentiation that involves perturbation of several nuclear transcription factors. We have established that thyroid hormone receptor beta1 (TRbeta1) is associated with the loss of TSHR gene expression in an anaplastic human thyroid cancer cell line, ARO. To demonstrate that TRbeta1 regulates TSHR gene expression, we performed electrophoresis mobility shift and 3,5,3'-triiodothyronine (T3) transactivation assays. As expected, TRbeta1 bound the synthesized oligomer containing TSHR promoter sequence by heterodimerizing with retinoid X receptor. When a chimeric reporter pTRCAT5'-146 enclosing the minimal TSHR promoter was applied for T3 transactivation assay, two TRbeta1-overexpressing transfectants of ARO cells (ARO1 and ARO2) demonstrated higher basal activity than their parental cells. Consequentially, T3 suppressed the reporter gene activity only in ARO1 and ARO2, but not in ARO cells. A point mutation creating a cAMP response element (CRE) in the reporter pTRCAT5'-146 CRE led to T3-induced suppression of the reporter gene in ARO cells without changing the basal or T3-induced activities in ARO1 and ARO2 cells. We conclude that the regulatory effect of T3 on TSHR gene expression is TR- and promoter DNA sequence-determined.
C Voigt, HP Holzapfel, S Meyer and R Paschke
G-protein-coupled receptor kinases (GRKs) are implicated in the pathophysiology of human diseases such as arterial hypertension, heart failure and rheumatoid arthritis. While G-protein-coupled receptor kinases 2 and 5 have been shown to be involved in the desensitization of the rat thyrotropin receptor (TSHR), their role in the pathophysiology of hyperfunctioning thyroid nodules (HTNs) is unknown. Therefore, we analyzed the expression pattern of the known GRKs in human thyroid tissue and investigated their function in the pathology of HTNs. The expression of different GRKs in human thyroid and HTNs was measured by Western blotting. The influence of GRK expression on TSHR function was analyzed by coexpression experiments in HEK 293 cells. We demonstrate that in addition to GRKs 2, 5 and 6, GRKs 3 and 4 are also expressed in the human thyroid. GRKs 2, 3, 5 and 6 are able to desensitize the TSHR in vitro. This GRK-induced desensitization is amplified by the additional over-expression of beta-arrestin 1 or 2. We did not find any mutations in the GRKs 2, 3 and 5 from 14 HTNs without TSHR mutations and Gsalpha mutations. The expression of GRKs 3 and 4 was increased in HTNs independently from the existence of TSHR mutations or Gsalpha mutations. In conclusion, the increased expression of GRK 3 in HTNs and the ability of GRK 3 to desensitize the TSHR in vitro, suggest a potential role for GRK 3 as a negative feedback regulator for the constitutively activated cAMP pathway in HTNs.