Ontogenesis of iodothyronine-5'-deiodinase. Induction of 5'- deiodinating activity by insulin, glucocorticoid, and thyroxine in cultured fetal mouse liver. K Sato, … , T Tsushima, K Shizume J Clin Invest. 1984;74(6):2254-2262. https://doi.org/10.1172/JCI111652. Research Article To elucidate the regulatory mechanism of ontogenetic development of iodothyronine-5'-deiodinase in the fetal and neonatal period, fetal mouse liver of the 19th day of gestation, in which no iodothyronine-5'-deiodinating activity was detectable, was cultured in Dulbecco-Vogt medium supplemented with 10% thyroid hormone-depleted fetal calf serum, insulin, hydrocortisone, and thyroid hormones. Iodothyronine-5'-deiodinating activity of the homogenate was assessed by the amount of iodide released from outer-ring-labeled reverse T3 and expressed as picomoles of 127I- per milligram of protein per minute. The enzyme activity was induced in a dose-dependent manner; optimal concentrations for insulin, hydrocortisone, and thyroxine were 1 microgram/ml, 0.4 microgram/ml, and 10(-6) M, respectively. Without supplementation of either hydrocortisone or thyroxine, no 5'-deiodination was detected. The enzyme activity was observed after 3 d of culture, peaked at days 14-20, and then gradually decreased. Lineweaver-Burk analysis revealed that the increase in activity was primarily due to an increase in Vmax (day 3, 0.2 pmol/mg protein per min; day 20, 2.5 pmol/mg protein per min). Half maximal thyroxine (T4) and triiodothyronine (T3) concentrations were 1 X 10(-7) M (free T4: 4 X 10(-10) M), and 2 X 10(-9) M (free T3: 5.0 X 10(-11) M), respectively, whereas reverse T3 did not elicit any activity at 10(-8)-10(-6) M. These results suggest that ontogenetic development of iodothyronine-5'-deiodinase in the liver of the fetal and […] Find the latest version: https://jci.me/111652/pdf Ontogenesis of lodothyronine-5'- Deiodinase Induction of 5'-Deiodinating Activity by Insulin, Glucocorticoid, and Thyroxine in Cultured Fetal Mouse Liver Kanji Sato, Hisoko Mimura, Doo Chol Han, Toshio Tsushima, and Kazuo Shizume Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, Ichigaya-Kawada-cho 10, Shinjuku-ku, Tokyo, Japan #162; Foundation for Growth Science in Japan, Shinjuku-ku, Tokyo, Japan Abstract. To elucidate the regulatory mecha- Half maximal thyroxine (T4) and triiodothyronine (T3) nism of ontogenetic development of iodothyronine-5'- concentrations were 1 X l0-7 M (free T4: 4 X 10-10 M), deiodinase in the fetal and neonatal period, fetal mouse and 2 X 10-9 M (free T3: 5.0 X 10-" M), respectively, liver of the 19th day of gestation, in which no iodothy- whereas reverse T3 did not elicit any activity at 10-8_ ronine-5'-deiodinating activity was detectable, was cul- 10-6 M. tured in Dulbecco-Vogt medium supplemented with These results suggest that ontogenetic development 10% thyroid hormone-depleted fetal calf serum, insulin, of iodothyronine-5'-deiodinase in the liver of the fetal hydrocortisone, and thyroid hormones. Iodothyronine- and neonatal mouse is induced by physiological concen- 5'-deiodinating activity of the homogenate was assessed trations of glucocorticoid and thyroid hormones, and by the amount of iodide released from outer-ring-labeled that insulin plays a permissive role in enhancing T3 reverse T3 and expressed as picomoles of 1271- per formation from T4 in the liver. milligram of protein per minute. The enzyme activity Introduction was in a dose-dependent manner; optimal con- induced Recently, the ontogenesis of thyroid hormone metabolism in centrations for insulin, hydrocortisone, and thyroxine peripheral tissues was extensively studied in man, sheep, chick, were 1 ;ig/ml, 0.4 jg/ml, and 10-6 M, respectively. and especially rodents (1-5). In contrast to newborn of humans Without supplementation of either hydrocortisone or and sheep, in which hypothalamic-pituitary-thyroid function thyroxine, no 5'-deiodination was detected. The enzyme is already mature and the sera contain adult thyroxine (T4)' activity was observed after 3 d of culture, peaked at days levels, the rat (and probably mouse) is delivered in the hypo- 14-20, and then gradually decreased. Lineweaver-Burk thyroid state. The serum T4 concentration of newborn of rats analysis revealed that the increase in activity was pri- is very low (1.6 ,ug/dl) and triiodothyronine (T3) is hardly marily due to an increase in Vmax (day 3, 0.2 pmol/mg detectable (-5 ng/dl) (1-3). Active thyroid hormone (T3) then protein per min; day 20, 2.5 pmol/mg protein per min). increases in parallel with serum T4. In fetal liver of mice (6) and rats (3), iodothyronine-5'-deiodinating activity is hardly detectable. However, in the neonatal period, there occurs a A part of this work was presented at the International Symposium for "Peripheral Metabolism of Thyroxine: Biochemical Background and progressive increase in the enzyme activity, which peaks at 2- Clinical Meaning," 15-18 May 1983, at the Reisensburg Castle, Ulm, 3 wk after birth in the case of mice (6). In parallel with the Germany. increase in enzyme activity, serum T3 increases progressively Address reprint requests to Dr. Sato, Department of Medicine, during the first month to a peak concentration of 60-90 ng/ Institute of Clinical Endocrinology, Tokyo Women's Medical College, dl (2, 3). The increase in T3 production in the peripheral Ichigaya-Kawadacho 10, Shinjuku-ku, Tokyo #162, Japan. tissues, especially in the liver, seems to be advantageous for Received for publication 10 February 1984 and in revised form 23 August 1984. 1. Abbreviations used in this paper: DTT, dithiothreitol; DV-T4(-)FCS, Dulbecco-Vogt medium supplemented with nonessential amino acids, J. Clin. Invest. penicillin, streptomycin sulphate, and 10% resin-treated fetal calf © The American Society for Clinical Investigation, Inc. serum; GSSG, glutathione disulfide; rT3, 3,3',5'-triiodo-L-thyronine; 0021-9738/84/12/2254/09 $ 1.00 3,3'-T2, 3,3'-diiodo-L-thyronine; T3, 3,3',5-triiodo-L-thyronine; T4, thy- Volume 74, December 1984, 2254-2262 roxine; T-GSH, total glutathione. 2254 Sato, Mimura, Han, Tsushima, and Shizume the survival of newborn mammals. However, the factors 30 and 40 mm. These floating loops were made of silicon tubing. A controlling this maturation process are not yet known. polyethylene shaft (2.5 X 8 mm) cut from a disposable micropipet tip On the assumption that iodothyronine-5'-deiodinase is (Exel tips for 20 Al, Shoei Co., Tokyo) was inserted into the tube to regulated by certain hormones, we developed an organ culture make a loop, which had been sterilized by autoclaving. Pieces of fetal liver were cultured at system of fetal mouse liver. We found that the ontogenetic 370C under 5% CO2 and 95% air for 9 d unless otherwise specified. Every 3 d, two thirds of the medium was replaced development of iodothyronine-5'-diodinase, which converts T4 with fresh medium. to T3 and reverse T3 (rT3) to 3,3'-T2 (7, 8), is induced by At the end of incubation, the membrane was washed with serum- glucocorticoid and thyroid hormones at their physiological free Dulbecco-Vogt medium; then, the membrane surface was scraped concentrations and that insulin plays a permissive role in with a sharp-edged spatula and hepatocytes that proliferated on the inducing the enzyme activity. membrane were transferred to a Potter-Elvehjem homogenizer. They were homogenized in 0.4 ml of ice-cold 0.25 M sucrose solution Methods containing 10 mM Tris-HCl buffer (pH 7.0) with a motor-driven teflon pestle at 3,000 rpm for 30 s. A 50-Ml aliquot of homogenate was [3',5'-'251]triiodo-L-thyronine (1251I-rT3) with a specific activity of about taken for measurement of protein by the method of Lowry et al. (12). 1 mCi/Mg was obtained from New England Nuclear Corp. (Boston, The homogenate was kept at 0-40C when deiodinating activity was MA). [3'-'25Iltriiodo-L-thyronine (125II-T3) and [3',5'-'23I]thyroxine ('2I- measured on the same day. Otherwise, it was frozen at -80'C until T4) with a specific activity of 1.5 mCi/Mg was prepared (9) and purified assayed. The enzyme activity was stable for at least 1 wk under this (10) as described elsewhere. Nonradioactive thyroxine (T4), 3,3',5- condition. triiodo-L-thyronine (T3), and hydrocortisone hemisuccinate were pur- Assay for iodothyronine-'-deiodinating activity. The enzyme activity chased from Sigma Chemical Co. (St. Louis, MO) and rT3 from was assessed as described elsewhere with slight modifications (13). In Calbiochem-Behring Corp. (San Diego, CA). Our T4 was contaminated brief, hepatocyte homogenates (10-100 Mg of protein) were incubated by 0.8% T3 (determined by radioimmunoassay), and used without in 200 Ml of sodium phosphate buffer (100 mM, pH 7.0) containing purification. AG-IX-10 anion exchange resin (chloride form, minus 10 mM DTT, 1.3 mM EDTA, and l0-7 M [3,5'-'251I]rT3. After 15- 400 mesh) and Dowex 50W-X4 cation exchange resin (200-400 mesh) min incubation under air, the reaction was stopped by adding 0.1 ml were obtained from Bio-Rad Laboratories (Richmond, CA). Fetal calf human serum followed by 1 ml of cold 10% TCA. After centrifugation serum and nonessential amino acids were obtained from Gibco Lab- at 3,000 rpm for 20 min, the supernatant was applied on Dowex 50W- oratories (Grand Island, NY) and other amino acids and vitamins for X4 cation exchange resin columns (200-400 mesh, 0.7 X 1.0 cm) tissue culture from Wako Pure Chemicals, Ltd. (Tokyo, Japan). equilibrated with 8 N acetic acid. Columns were washed twice with 1 Dithiothreitol (DTT) was purchased from Nakarai Chemicals, Ltd.