Cells respond rapidly to endoplasmic reticulum (ER) stress by blocking protein translation, increasing protein folding capacity, and accelerating degradation of unfolded proteins via ubiquitination and ER-associated degradation pathways. The type II iodothyronine deiodinase (D2) is a type I endoplasmic reticulum (ER)-resident thioredoxin fold-containing selenoprotein that activates thyroid hormone. D2 plays critical roles in mammalian development, energy homeostasis and hypothalamic-pituitary-thyroid axis feedback mechanism. A common polymorphism in the gene encoding the activating deiodinase (Thr92Ala-D2) is known to be associated with quality of life in millions of patients with hypothyroidism. A prevalent Thr92Ala-D2 polymorphism has been identified and results in a single amino change at position 92 within an 18 amino acid loop that controls D2 ubiquitination for proteasomal destruction. Hypothyroid individuals carrying this polymorphism were found to prefer a therapy that includes T3 (triiodothyronine) vs monotherapy with L-T4 (levothyroxine) alone, suggesting defective Ala92-D2 catalysis. In addition, the Thr92AlaD2 polymorphism has been associated with conditions aside from symptomatic hypothyroidism such as mental retardation, low IQ, and bipolar disorder; this supports the hypothesis that Ala92-D2-expressing is disruptive aside from impaired T4 (thyroxine) activation. This polymorphism results in a single amino acid change within the D2 molecule where its susceptibility to ubiquitination and proteasomal degradation is regulated. The aim of this project is to evaluate the mechanisms involved in animals induced to metabolic alterations and with or without D2 polymorphism. For this purpose, mice will be induced to obesity with a high fat diet for 2 weeks to induce insulin resistance and ER stress. After this, D2 activity will be measured and correlated with markers of ER stress, i.e. gene expression (mRNA expression of UPR proteins: GADD34, CHOP, GRP78/BIP, XBP1, ATF4 and ATF6) and protein expression and phosphorylation level (eIF2a, Ire1 and PERK) of UPR pathway and proteasome activity in different areas of the brain (hypothalamus, hippocampus, cerebral cortex). In parallel, we will investigate the role of oleic- or palmitic acid-induced ER stress in D2 activity in cells stably expressing D2 or the polymorphic AlaD2. This study may contribute to the understanding of the involvement of the ER stress in hypothyroid disorders under metabolic conditions.
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