**3. The role of DIO1 and DIO2 deiodinase enzymes in thyroid hormone production of Graves' hyperthyroidims**

Hyperthyroidism is characterized by increased serum FT4 and FT3 levels, which can be associated with Graves' Disease, toxic goiter, destruction-induced thyrotoxicosis and subacute thyroiditis. Thyroid follicular cells possess both DIO1 and DIO2 enzymes, but not DIO3 enzyme. The amount of produced FT4 and FT3, and the ratio of FT3 to FT4 can help with the diagnosis [32]. Serum FT3 levels are predominant and are better formed than FT4 in hyperthyroidism connected to Graves' Disease or toxic goiter [33]. In Graves' hyperthyroidism, the increase in the daily production of T3 and T4 was 7-fold and 3.5-fold, respectively. Laurberg and coworkers demonstrated that the major source of excess T3 derived from increased thyroidal DIO1 and DIO2 activities (in a ratio of 3 to 1). This is in contrast to what is found in euthyroidism, where 20% of T3 came from thyroidal production and 80% from extrathyroidal deiodination [25]. In hyperthyroidism, a large part of T3 levels was produced by the thyroid (in 57–77%) by way of converting T4 to T3 with decreased peripheral deiodination. The extrathyroidal DIO2 activities were decreased in hyperthyroidism with the exception of the thyroidal one due to the increased thyroidal formation of T4 and T3. Maia and coworkers supported that thyroidal DIO1 activity is responsible for 67% of T3 production in hyperthyroidism [22]. In HEK 293 cells, which transiently expressed DIO1 and DIO2 enzymes, the effect of 2–20-200 pM T4 was studied on these cells modeling hypo-, eu- and hyperthyroid states. DIO1 activity was continuous, but DIO2 activity was decreased by the concentration of 200 pM T4. Salvatore and coworkers emphasized the greater role of DIO2 enzyme in the excess T3 in Graves' hyperthyroidism [34]. Ito and coworkers suggested that thyroidal DIO1 and specifically, DIO2 could be contributed to the higher ratio of FT3 to FT4 in Graves' hyperthyroidism [35]. The lower ratio of T3 to T4 can help us with the diagnosis of destruction-induced thyrotoxicosis and subacute thyroiditis [36]. Values less than of 20 confirm the above mentioned diseases, while the values above 20 are connected to Graves' hyperthyroidism. Weetman and coworkers made the DIO1 and DIO2 activities responsible for the syndrome of low T4 with increased T3 levels during PTU treatment [37]. Thyroidal DIO1 activity is mainly regulated by cAMP at pretranslational levels, similarly to TSH receptor stimulating antibody-induced cAMP. Thyroglobulin and iodine contents of thyroid can influence the generation of T4 and T3 through the rate of hydrolysis from the colloid-embedded thyroglobulin. This condition can contribute to the alterations in the production of thyroid hormones. Very few reports could be found, which explained in detail the thyroid hormone production connecting to the formation of the coupling mechanism alone or together with deiodinase conversion. Iodide alone inhibited both thyroidal deiodinase activities rapidly decreasing the circulating T3 by 50% and T4 by 70% [33]. Ipodate is also a potent inhibitor for DIO1 and DIO2 enzymes due to its iodine content of 64%. Ipodate with PTU resulted in a profound decrease in serum T3. In untreated Graves' hyperthyroidism, the T3 content of Tg was 2-fold of what was found in euthyroidism [38]. In hyperthyroidism, local DIO2 activity is required for the intrapituitary production of T3, which is responsible for the acute decrease in TSH levels [39].

## **4.** *In vitro* **model for the measurement of tissue-specific DIO2 activities**

In our study, homogenized (supernatant of 100 000 x g separated by centrifugations) thyroidal, skeletal and eye muscle tissue fractions, called cytosol fractions were applied for the measurements of deiodinase enzyme activities [40]. Thyroid

#### *Deiodinase Enzymes and Their Activities in Graves' Hyperthyroidism DOI: http://dx.doi.org/10.5772/intechopen.97007*

tissues were obtained from the removal of euthyroid goiter; the removal of skeletal muscle during accident surgery and the removal of extraocular muscle during strabismus surgery. All tissue fractions contained DIO2 enzyme, the activity of which was measured in the presence of patient sera with Graves' Disease with respect to the different thyroid hormonal stages. The DIO2 content of cytosol fractions was proofed before the study using guinea pig sera immunized with TCSS and LVFR peptides. Both peptides were corresponding to amino acid sequences of human DIO2 (GenBank AAD45494–1) and contained the selenocysteine at position 133 in the active center of the enzyme: *LVVNFGSATCPPFTSQLPAFRKLVEEFSS.* TCSS peptide (aa 132–152): T**CC**PP**TF**SQLPAFRKLVEEFSS was synthesized with double cysteins replaced at position 133, as well as amino acids reserved at positions 136 and 137. LVFR peptide (aa 124–144): LVVNFGSAT**C**PPFTSQLPAFR was 100% identical to the original amino acid sequence. The bindings of immunized sera to cytosol fractions and to tissue sections were investigated with enzymelinked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Immunized sera against TCSS peptide resulted in more intensive positive bindings to the cytosol fractions and gave positive reactions to tissue sections.

The patient sera of hyper-, eu- and hypothyroid Graves' Disease were added to thyroidal, skeletal and eye muscle cytosol fractions, which contained DIO2 enzyme activities. The study could be considered as an *in vitro* model, in which the patient sera included the actual hormonal and autoantibody parameters. The effects of these parameters were measured on DIO2 activities. The results, after evaluating them with respect to the parameters, may contribute to gaining useful data for the course and treatment of disease. Thyroidal DIO1 activities were inhibited by 2 μM PTU. The sample mixture contained 12.5 μg protein per cytosol fraction. Radioiodine labeled T4 (125I-T4) 1 kB/50 μl was the substrate in reducing condition [20 mM dithiothreitol (DDT)]. The results were extrapolated at 1 pmol/ T4 of patient serum. DIO2 enzyme activity was expressed as pmol of T4 converted per mg/min of protein. The whole protocol is described in detail in our previous paper [41].
