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SUMMARY The physiological role played by thyroid hormones (TH) in hydro-osmotic homeostasis in fish remains a controversial issue. Previous studies have shown that in Fundulus heteroclitus (killifish) hypo-osmotic stress increases liver iodothyronine deiodinase type 2 (D2) mRNA and D2 activity. In this study we identified two conserved osmotic response element (ORE) motifs in the promoter region of the killifish D2 gene (FhDio2) and examined their possible role in the transcriptional regulation of FhDio2during hypo-osmotic stress. As assessed by the electrophoretic mobility shift assay, results from in vivo and in vitro experiments demonstrate that exposure to an abrupt hyposmotic challenge triggers in the liver of killifish a strong nuclear recruitment of a putative osmotic response element binding protein (OREBP). This protein–DNA binding is time-dependent, attains a maximum within 2–8 h after the osmotic stress,and is followed by a significant increase in D2 activity. Furthermore,protein–DNA binding and the subsequent elevation in enzyme activity were blocked by the tyrosine kinase inhibitor genistein. Thus, during hypo-osmotic stress, a putative OREBP kinase-activated pathway stimulates FhDio2transcription and enzymatic activity. These data and the fact that D2 is the major enzyme providing local intracellular T3 suggest that TH plays a direct role in osmoregulation in fish, possibly by participating in hepatic ammonia metabolism. This study provides important insight into the physiological role of TH in hydro-osmotic homeostasis in fish.

Substrate availability has been thought to be a major regulator of the outer-ring deiodinating pathway (ORD) in fish. However, current information strongly suggests that while fish iodothyronine deiodinase type 2 (D2) responds to iodothyronines in the same manner as its mammalian counterpart, fish deiodinase type 1 (D1) exhibits a distinct response. Furthermore, 3,5-T 2 , generally considered to be an inactive product of iodothyronine metabolism, has recently been described as bioactive, but its effects upon D1 and D2 are not yet known. We examined the effect that short-term immersion in T 4 , T 3 , and 3,5-T 2 (0.1 μM; 12 or 24 h) exerts on both D1 and D2 activities and on the levels of expression of D1 and D2 mRNAs in killifish liver. In agreement with previous reports in teleosts, no iodothyronine exerted a significant effect on D1 enzymatic activity. However, all three iodothyronines significantly decreased D2 activity. Furthermore, at 24 h post-immersion T 4 , T 3 , and 3,5-T 2 inhibited both D1 and D2 transcription. Together, the present results confirm the differential effect of iodothyronines upon the hepatic ORD pathway in fish and show that this effect can occur at a transcriptional level. Furthermore, we provide the first evidence that 3,5-T 2 can affect both activity and transcription of hepatic deiodinases in teleosts.

Until recently, 3,5-diiodothyronine (3,5-T2) has been considered an inactive by-product of triiodothyronine (T3) deiodination. However, studies from several laboratories have shown that 3,5-T2has specific, nongenomic effects on mitochondrial oxidative capacity and respiration rate that are distinct from those due to T3. Nevertheless, little is known about the putative genomic effects of 3,5-T2. We have previously shown that hyperthyroidism induced by supraphysiological doses of 3,5-T2inhibits hepatic iodothyronine deiodinase type 2 (D2) activity and lowers mRNA levels in the killifish in the same manner as T3and T4, suggesting a pretranslational effect of 3,5-T2(Garcia-G C, Jeziorski MC, Valverde-R C, Orozco A. Gen Comp Endocrinol 135: 201–209, 2004). The question remains as to whether 3,5-T2would have effects under conditions similar to those that are physiological for T3. To this end, intact killifish were rendered hypothyroid by administering methimazole. Groups of hypothyroid animals simultaneously received 30 nM of either T3, reverse T3, or 3,5-T2. Under these conditions, we expected that, if it were bioactive, 3,5-T2would mimic T3and thus reverse the compensatory upregulation of D2 and tyroid receptor β1 and downregulation of growth hormone that characterize hypothyroidism. Our results demonstrate that 3,5-T2is indeed bioactive, reversing both hepatic D2 and growth hormone responses during a hypothyroidal state. Furthermore, we observed that 3,5-T2and T3recruit two distinct populations of transcription factors to typical palindromic and DR4 thyroid hormone response elements. Taken together, these results add further evidence to support the notion that 3,5-T2is a bioactive iodothyronine.