Life Sciences 116 (2014) 74–82

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Effects of triiodothyronine on turnover rate and metabolizing enzymes for thyroxine in thyroidectomized rats Hidenori Nagao a,⁎, Makoto Sasaki a, Tetsuya Imazu a, Kenjo Takahashi b, Hironori Aoki a, Kouichi Minato a a b

Pharmacokinetics Research Department of ASKA Pharmaceutical Co., Ltd., Kawasaki, Japan Department of Analytical Research, ASKA Pharma Medical Co., Ltd., Kawasaki, Japan

a r t i c l e

i n f o

Article history: Received 11 June 2014 Accepted 13 September 2014 Available online 28 September 2014 Keywords: Thyroid hormones Isotope tracer On-line SPE LC-MS/MS Turnover rate Thyroidectomy Deiodinases UDP-glucuronosyltransferases Sulfotransferases

a b s t r a c t Aim: Previous studies in rats have indicated that surgical thyroidectomy represses turnover of serum thyroxine (T4). However, the mechanism of this process has not been identified. To clarify the mechanism, we studied adaptive variation of metabolic enzymes involved in T4 turnover. Main methods: We compared serum T4 turnover rates in thyroidectomized (Tx) rats with or without infusion of active thyroid hormone, triiodothyronine (T3). Furthermore, the levels of mRNA expression and activity of the metabolizing enzymes, deiodinase type 1 (D1), type 2 (D2), uridine diphosphate-glucuronosyltransferase (UGT), and sulfotransferase were also compared in several tissues with or without T3 infusion. Key findings: After the T3 infusion, the turnover rate of serum T4 in Tx rats returned to normal. Although mRNA expression and activity of D1 decreased significantly in both liver and kidneys without T3 infusion, D2 expression and activity increased markedly in the brain, brown adipose tissue, and skeletal muscle. Surprisingly, hepatic UGT mRNA expression and activity in Tx rats increased significantly in comparison with normal rats, and returned to normal after T3 infusion. Significance: This study suggests that repression of the disappearance of serum T4 in rats after Tx is a homeostatic response to decreased serum T3 concentrations. Additionally, T4 glucuronide is a storage form of T4, but may also have biological significance. These results suggest strongly that repression of deiodination of T4 by D1 in the liver and kidneys plays a major role in thyroid hormone homeostasis in Tx rats, and that hepatic UGT also plays a key role in this mechanism. © 2014 Elsevier Inc. All rights reserved.

Introduction Pro-thyroid hormone, thyroxine (T4) is the main secretory product of the thyroid gland, and serum T4 is primarily metabolized by deiodinases and conjugation enzymes. Deiodinases consist of three types. Type 1 deiodinase (D1) catalyzes both outer ring deiodination (ORD) and inner ring deiodination (IRD) in the liver, kidney, pituitary gland, and thyroid (Chopra, 1977; Green, 1978). Type 2 deiodinase (D2) only catalyzes ORD in the central nervous system, brown adipose tissue (BAT), and skeletal muscle (Silva et al., 1982; Silva and Larsen, 1983; Marsili et al., 2010). The ORD of T4 is the only way to produce active thyroid hormone, triiodothyronine (T3) and is an important activating pathway. Biological activity of T3 is three to four times more potent than that of T4. Type 3 deiodinase (D3) only catalyzes IRD in the brain, skin, and placenta (Kaplan et al., 1983; Huang et al., 1985). This enzyme catalyzes the conversion of T4 to inactive reverse T3 (Gereben et al., 2008). ⁎ Corresponding author at: Pharmacokinetics Research Department of ASKA Pharmaceutical Co., Ltd., 5-36-1, Shimosakunobe, Takatsu-ku, Kawasaki 213-8522, Japan. Tel.: +81 44 812 8656; fax: +81 44 822 1265. E-mail address: [email protected] (H. Nagao).

http://dx.doi.org/10.1016/j.lfs.2014.09.016 0024-3205/© 2014 Elsevier Inc. All rights reserved.

Conjugation of the phenolic hydroxyl group with glucuronic acid or sulfuric acid is an important metabolic pathway. Both conjugates are considered to be biologically inactive and have increased water solubility, facilitating urinary and biliary excretion. Uridine diphosphateglucuronosyltransferases (UGTs) are classified into two families, UGT1 and UGT2, in both rats and humans (Tukey and Strassburg, 2000). In rat UGT1 family, UGT1A1 and UGT1A6 are important isoforms responsible for the glucuronidation of T4 in liver and kidneys (Vansell and Klaassen, 2002). Sulfotransferases (SULTs) consist of three families, phenol SULTs, estrogen SULTs, and hydroxysteroid SULTs (Weinshilboum et al., 1997; Falany, 1997). Sulfation of iodothyronines is catalyzed by phenol SULTs (SULT1A1, 1B1, and 1C1) in the liver and kidneys (Dunn and Klaassen, 2000). In male rat liver, SULT1C1 is almost exclusively expressed (Liu and Klaassen, 1996; Yamazoe et al., 1994). We previously reported that serum T4 and T3 levels in rats were decreased but not completely abolished after surgical Tx. These hormone levels were maintained at a constant very low level throughout the experimental period, and the turnover rate of T4 in Tx rats was slower than in normal rats (Nagao et al., 2011). These findings suggest that the disappearance of serum T4 is suppressed by Tx, and serum T4 is supplied by extra-thyroidal tissues (e.g. secretion of extra-thyroidal storage, enhancement of enterohepatic recirculation, and production

H. Nagao et al. / Life Sciences 116 (2014) 74–82

in extra-thyroidal tissues). In contrast, although serum T4 levels of iodine-deficient rats are decreased similar to in Tx rats, T3 levels are normal (Minato et al., 2012). Furthermore, there were no differences in turnover rates of T4 among iodine-deficient and normal rats. These findings suggested that prolongation of the half-life of serum T4 after Tx is a homeostatic response to stabilize thyroid hormone activity to decreased serum T3 concentrations. However, the homeostatic mechanisms of thyroid hormone activity are complex, and many details remain unknown. The aim of the present study was to clarify the mechanism of repression of serum T4 degradation after Tx. We evaluated turnover rates of serum T4 in Tx rats infused with T3 using a stable isotopelabeled tracer ([13C9]T4) method (Nagao et al., 2011). Furthermore, we investigated the mRNA expression and activity of T4 metabolizing enzymes (D1, D2, UGTs, and SULTs) in several tissues in Tx rats infused with or without T3. Materials and methods Chemicals T4, T3 and reverse T3 (rT3) were purchased from Sigma-Aldrich Co. (NJ, USA). L-Thyroxine-[L-tyrosine-2H5]HCl ([2H5]T4) was purchased from IsoSciences, LLC (King of Prussia, PA, USA). [13C9] T4 was synthesized chemically from [13C9] tyrosine in our laboratory (Nagao et al., 2011). [125I]T4 and [125I]rT3 were obtained from PerkinElmer (MA, USA). They were purified on Sephadex LH-20 (Pharmacia, Uppsala, Sweden) just before each assay (Rutgers et al., 1989). All other chemicals and reagents were of the highest analytical grade commercially available.

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(final concentration; 25 μg/ml in 0.5 mM NaOH/saline) was diluted after NaOH solubilization (5 mg T3/20 μl of 1 M NaOH). Five sham-operated (control) and Tx rats received pumps with 0.5 mM NaOH/saline only. On day 14 after osmotic minipump implantation, [13C9]T4 was administered intravenously to all group rats at a dose of 1.5 μg/500 μl/kg (Nagao et al., 2011). On the day before pump implantation (day −1), day 13, and days 14–23 (for 10 days after [13C9]T4 injection), serum samples were collected from tail vein. All samples were obtained between 9 and 11 a.m. to minimize fluctuations in thyroid hormones (Jeremiah et al., 1990) and stored at −20 °C until analyzed. Experiment 2 Osmotic minipumps, delivering 1.5 μg T3/head/day (Tx + T3 rats; n = 6) were implanted subcutaneously under isoflurane anesthesia. Six control and Tx rats received pumps with 0.5 mM NaOH/saline only. On day 14 after the implantation, all rats were exsanguinated via a needle in the abdominal aorta. The liver, kidneys, brain, brown adipose tissue (BAT), and thigh muscles (skeletal muscles) were dissected and processed immediately frozen in liquid nitrogen and stored at −80 °C until measured for enzyme activity. Aliquots of these tissues were stored at − 20 °C in Allprotect tissue reagent (QIAGEN, Venlo, Netherlands) until analyzed for RT-PCR. Serum thyroid hormone concentrations

Seven-week-old male Sprague–Dawley rats were obtained from Charles River Laboratories Japan (Kanagawa, Japan). Animals were fed a commercial diet (FR-2, Funabasi Farm Co., Chiba, Japan) and filtered tap water ad libitum. The cages were located in a light (0800–2000 h lights on), temperature (23 ± 5 °C) and humidity (60 ± 20%) controlled room. The rats were allowed to acclimatize for 1 week before starting the experiments. All experimental procedures were approved by the Animal Research Committee of ASKA Pharmaceutical Co., in accordance with the Basic Guidelines for Proper Conduct of Animal Testing and Related Activities in the Research Institutions under the Jurisdiction of the Ministry of Health, Labour and Welfare of Japan.

T3, T4, and [13C9]T4 were determined by the method using on-line SPE LC-MS/MS (Nagao et al., 2011). In brief, an API5000 triple–quadrupole mass spectrometer (AB SCIEX, CA, U.S.A.) equipped with a TurboIonSpray source and Shimadzu HPLC system was employed to perform the analysis using isotope dilution with deuterium labeled internal standard, [2H5]T4 . A 20 μl aliquot of rat serum was mixed with internal standard acetonitrile solution for deproteinization, and was diluted with 0.1 vol.% formic acid. After centrifugation, the supernatant was injected into the LC-MS/MS system. On-line extraction and chromatographic separation of the analytes were performed using respectively a Shim pack MAYI-ODS, 2.0 mm i.d. × 10 mm, 50 μm (Shimadzu, Kyoto, Japan) and a Synergi Polar-RP 80A, 2.0 mm i.d. × 50 mm, 4 μm (Phenomenex, Utrecht, the Netherlands). Details of the LC conditions including gradient elution and the selected reaction monitoring transitions are described in a previous report (Nagao et al., 2011). Linear calibration curves of T3, T4, and [13C9] T4 were obtained in the concentration range of 0.1–100 ng/ml, with a lower limit of quantitation of 0.1 ng/ml.

Thyroidectomy

Tissue preparations

Rats were made hypothyroid by surgical thyroidectomy as described previously in detail (Nagao et al., 2011). After the surgery, serum TSH rapidly increased and, body weight gain was complete stasis. Complete resection of the thyroid in the Tx rats was confirmed at the end of the experiment by macroscopic observation at necropsy. In addition, serial sections from tracheal tubes (area of thyroid glands in Tx rats) were reviewed by pathologists and thyroid tissues including follicular epithelial cells were not observed.

Frozen tissues (liver, kidneys, brain, BAT and skeletal muscles) were pulverized, and the obtained powder was suspended in 5 volumes icecold preparation buffer (20 mM Tris–HCl(pH7.6), 0.25 mM sucrose, 1.2 mM EDTA, 5 mM dithiothreitol (DTT, Wako Pure Chemical Industries, Osaka, Japan)), and complete protease inhibitor cocktail (Roche, Basel, Switzerland). Homogenization was accomplished using a Tissue Tearor (BiospecProducts, OK, USA). The homogenates were centrifuged at 1000 ×g at 4 °C for 15 min to remove any remaining particle, and stored at −80 °C until analyzed except the skeletal muscles. The homogenate of the skeletal muscles was used for the following process without freeze preservation. Aliquots of the homogenates of liver, kidneys and skeletal muscles were centrifuged at 10,000 ×g at 4 °C for 20 min. The supernatants were centrifuged at 105,000 ×g at 4 °C for 1 h, and the microsomal pellets of the liver and kidneys were resuspended in ice-cold preparation buffer. Microsomes (liver and kidneys) and cytosol (skeletal muscles) were stored at − 80 °C until analyzed. Protein levels of tissue fractions were measured by the method of Lowry et al. (1951), using BSA as a reference.

Experimental animals

Experiment 1 At 3 weeks after Tx treatments, 15 Tx rats were divided into 3 groups (n = 5 each). Osmotic minipumps (Alzet, model 2ML4, DURECT, CA, USA), delivering 1.5 μg T3/head/day (Tx + T3 rats; n = 5) or 7.5 μg T3/head/day (Tx + HT3 rats; n = 5) were implanted subcutaneously on day 0 under isoflurane anesthesia. The doses of T3 were determined by our preliminary experiment and previous literature (Nguyen et al., 1993). The vehicle was saline, in which T3

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Enzyme activity assays Deiodinases Assays for deiodinase activity were performed essentially using the slightly modified methods (Visser et al., 1988; Richard et al., 1998; Marsili et al., 2010). In brief, D1 activity was assayed in liver and kidney homogenates (0.3 mg protein/ml) using 400 nM (100,000 cpm) [125I] rT3 and 10 mM DTT, and incubated for 60 min at 37 °C. D2 activity was assayed in brain and BAT homogenates (1 mg protein/ml), and skeletal muscles microsomes (2 mg protein/ml) using 1 nM (100,000 cpm) [125I]T4 and 25 mM DTT in the presence of 1 mM propyl-2-thiouracil (Sigma, NJ, USA) and 1 μM T3, and incubated for 60 min (brain and BAT) or 16 h (skeletal muscles) at 37 °C. These reactions were stopped by addition of 100 μl 5% ice-cold BSA followed by addition of 500 μl 10% trichloroacetic acid for deproteinization. Samples were centrifuged at 1500 ×g at 4 °C for 15 min and the released 125I− was purified from the supernatant on Sephadex LH-20 mini-columns, which had been equilibrated and eluted with 0.1 M HCl.

UGTs Hepatic and renal T4-UGT enzyme assays were carried out essentially using the slightly modified method of Beetstra et al. (1991). In brief, microsomes (1 mg protein/ml) were incubated for 60 min at 37 °C with 1 μM (100,000 cpm) [125I]T4 in a total volume of 200 μl incubation medium (in a final concentration of 75 mM Tris–HCl, 7.5 mM MgCl2, 0.025% alamethicin (Sigma), 1 mM propyl-2-thiouracil (PTU) at pH 7.4) in the presence or absence of 5 mM uridine diphosphoglucuronic acid (Sigma). Reactions were stopped by the addition of 0.8 ml ice-cold 0.1 M HCl. After centrifugation, the supernatant was analyzed for glucuronide formation by chromatography on Sephadex LH-20 mini-columns.

SULTs Hepatic and renal T4-SULT enzyme assays were carried out essentially using the slightly modified method of Kaptein et al. (1997). In brief, cytosols (0.1 mg protein/ml) were incubated for 30 min at 37 °C with 0.1 μM (100,000 cpm) [125I]T4 in a total volume of 200 μl incubation medium (in a final concentration of 100 mM phosphate, 2 mM EDTA, 1 mM PTU at pH 7.2) in the presence or absence of 50 μM 3′-phosphoadenosine 5′-phosphosulfate (Sigma). Reactions were stopped by the addition of 0.8 ml ice-cold 0.1 M HCl. After centrifugation, the supernatant was analyzed for sulfate formation by chromatography on Sephadex LH-20 mini-columns.

RT-PCR Total RNA was isolated from liver, kidneys, brain (cerebral cortex), BAT and skeletal muscles using Qiazol lysis reagent (QIAGEN, Venlo, Netherlands) and subsequently purified using RNeasy mini kit (QIAGEN) following the manufacturer's instructions. Total RNA was reverse-transcribed using QantiTect Reverse Transcription Kit (QIAGEN). Real-time PCR was performed using a thermal cycler, LightCycler Quick System 350S (Roche Diagnostics, Mannheim, Germany), with LightCycler FastStart DNA MasterPLUS SYBR Green I (Roche Diagnostics) and 200 nM of each primer as listed Table 1. The primers were purchased from NIHON GENE RESEARCH LABORATORIES INC. (Miyagi, Japan). Non-template and water controls were used to detect non-specific amplification. Relative expression values were calculated based on the standard curve with LightCycler3 Data Analysis software version 3.5.28 (Roche Diagnostics) using a 1:10 dilution series of pooled cDNA as the standard. The reference gene was used for normalization: glyceraldehyde-3-phosphate dehydrogenase (Gapdh).

Table 1 Primer sequences for the quantitative RT-PCR analyses. Gene

Sequence

D1

Forward: TGG TGG ACA CAA TGC AGA Reverse: TCC AAG GGC CAG GTT TAC D2 Forward: CTG ACC TCA GAA GGA CTA CG Reverse: GCT TCA GGA TTG GAC ACG Ugt1a1 Forward: CTT CCG TGT GGC TCC ATT Reverse: ATG TTC TTC ACC CGC TGC Ugt1a6 Forward: TGT GAT CCT GGC TGA GTA Reverse: GGG AAA TGT CAT GTG GTC Sult1a1 Forward: GCC ATT GCA GAA CTT CAC AG Reverse: CAC ACT TCT CTA GCT TGC CA Sult1c1 Forward: CAA CGG GCC AAC ACC TAT Reverse: GCA TTT TGT TAG CCA GAT CCA Gapdh Forward: TGA CAA CTT TGG CAT CGT Reverse: ATG CAG GGA TGA TGT TCT

Locationa TM (°C)

Accession no.b

596–614 671–689 163–183 246–264 482–500 625–643 550–568 665–683 152–172 237–257 349–367 398–419 565–583 663–681

85.0

NM_021653

83.8

NM_031720

86.8

NM_012683

84.7

NM_057105

83.7

NM_031834

83.0

NM_031732

87.2

NM_017008

a Locations of the primers are based on the National Center for Biotechnology Information (NCBI) reference sequence. b NCBI reference sequence number.

Kinetic analysis The methods used to determine turnover rate and half-life of T4 were described in detail in a previous publication (Nagao et al., 2011). Statistical analysis Differences between the groups were assessed by one way analysis of variance using the SAS System Release 9.2 (SAS Institute Inc., Cary, NC, USA). Post hoc testing was performed for inter-group comparisons using the Tukey's test. Values are statistically significant at p b 0.05. The results were expressed as the mean ± S.D. Results Serum concentrations of thyroid hormones Serum T3 and T4 concentrations were determined before (day −1) and after (day 13) subcutaneous implantation of osmotic minipumps, delivering vehicle (control and Tx), 1.5 μg T3/head/day (Tx + T3) or 7.5 μg T3/head/day (Tx + HT3) to sham-operated or Tx rats (Fig. 1). In the all Tx groups before implantation, serum T3 and T4 levels were significantly decreased compared with control group (F(3,16) = 375.8, p b 0.01 and F(3,16) = 134.0, p b 0.05). These levels in the Tx group were not significantly changed between before and after vehicle infusion. In the Tx + T3 and Tx + HT3 groups after T3 infusion, serum T3 levels significantly increased compared with each group before infusion (F(1,8) = 674.38, F(1,8) = 41.5, both p b 0.01), and were approximately 100% and 170% those of control group, respectively. Moreover, serum T4 levels in both groups after T3 infusion significantly decreased by approximately 50% compared with before T3 infusion, respectively (F(1,8) = 22.2, p b 0.01 and F(1,8) = 10.5, p b 0.05). Effects of T3 replacement on serum T4 turnover in Tx rats To investigate the effects of T3 replacement on serum T4 turnover, serum [13C9]T4 concentrations were determined after a single intravenous administration of [13C9]T4 (1.5 μg/kg) to four groups of rats (control, Tx, Tx + T3, and Tx + HT3) (Fig. 2, Table 2). In Tx rats, serum [13C9]T4 concentrations gradually decreased compared with control rats after administration of [13C9]T4, and the turnover rate (kel) was 0.67 ± 0.07 day−1, approximately 2-fold slower than in control rats (1.16 ± 0.11 day− 1), and the half-life (t1/2) was approximately 1.1 days. These results were consistent with our previous report

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Table 2 Turnover rates and half-lives of serum T4 in Control and Tx rats subcutaneously infused with vehicle or T3. Control

Tx

Tx + T3

Tx + HT3

T3 dosea

0

0

1.5

7.5

kel (day−1) t1/2 (day)

1.16 ± 0.11 0.60 ± 0.06

0.67 ± 0.07** 1.05 ± 0.11**

1.27 ± 0.18## 0.56 ± 0.08##

1.15 ± 0.14 0.61 ± 0.08

Data are expressed as the mean values ± S.D. (n = 6). **p b 0.01 compared with the controls. ## p b 0.01 compared with the Txs. kel, Turnover Rate; t1/2, Half-Life; Tx, Thyroidectomy. a The dose are in micrograms per head/day.

Fig. 1. Serum thyroxine (T4) and triiodothyronine (T3) levels before and after subcutaneous implantation of vehicle or T3 in thyroidectomized (Tx) rats. Tx rats were infused with (A) vehicle, (B) T3 (1.5 μg/head/day), and (C) T3 (7.5 μg/head/day). Serum samples were collected before pump implantation (day − 1), and after day 13. T4 and T3 levels are expressed relative to the average level in the controls. All data represent the mean ± S.D. of five animals. *p b 0.05 and **p b 0.01 compared with the controls. #p b 0.05 and ## p b 0.01 compared with the before infusions.

(Nagao et al., 2011). In contrast, the turnover rate and t1/2 in Tx + T3 and Tx + HT3 rats returned to normal values (approximately 1.2 day−1 and 0.6 days).

Effects of T3 replacement on T4-metabolizing enzymes in Tx rats To further investigate the mechanism of repression of serum T4 degradation by Tx treatment, mRNA expression and activity of T4metabolizing enzymes were determined in liver, kidneys, brain, BAT, and skeletal muscles. For RT-PCR analysis, the observed linearity was good for the standard curve of each target cDNA, and the correlation coefficients were shown each figure.

Hepatic and renal D1 activity (Table 3) was significantly decreased in Tx rats compared with control rats (F(2,16) = 62.2, p b 0.01 and F(2,16) = 410.1, p b 0.01): 30% for liver and 18% for kidneys (Fig. 3A and B). The relative abundance of D1 mRNA was also significantly decreased in both tissues in Tx rats (F(2,15) = 31.9, p b 0.01 and F(2,13) = 28.3, p b 0.01) (Fig. 3C and D). In Tx rats infused with T3, D1 activity and mRNA expression were significantly increased in liver and kidneys compared with Tx rats (p b 0.05 for liver and kidneys activity, and p b 0.01 for liver and kidneys mRNA), and returned to control levels. D2 activity in the brain, BAT, and skeletal muscles was significantly increased in Tx rats compared with control rats (F(2,16) = 66.6, p b 0.01, F(2,15) = 23.9, p b 0.01, and F(2,15) = 8.5, p b 0.05): 462% for brain, 3055% for BAT, and 186% for skeletal muscle (Fig. 4A, B and C). D2 mRNA levels were also significantly increased in brain and BAT of Tx rats (F(2,14) = 4.0, p b 0.05 and F(2,13) = 3.0, p b 0.05) (Fig. 4D and E). Skeletal muscle D2 mRNA was slightly increased in Tx rats, but this difference was not statistically significant (Fig. 4F). However, when serum T3 levels in Tx rats were returned to normal by T3 replacement, D2 activity and mRNA expression were not normalized. Although BAT D2 activity was significantly decreased compared with Tx rats (p b 0.01), brain and skeletal muscle D2 activity did not change. Hepatic T4-UGT activity in Tx rats significantly increased to 200% of control rats (Table 3, F(2,14) = 15.3, p b 0.05), but returned to normal levels after T3 replacement (Fig. 5A). These changes paralleled those of hepatic Ugt1a1 mRNA levels (F(2,14) = 9.8, p b 0.01), but not Ugt1a6 mRNA levels (Fig. 6A and B). Although renal Ugt1a1 and Ugt1a6 mRNA levels were slightly changed by Tx treatment or T3 replacement (Fig. 6C and D), T4-UGT activity did not change (Table 3, Fig. 5B). Hepatic and renal T4-SULT activity did not significantly change in any group (Fig. 7). Moreover, mRNA expression of Sult1a1 and Sult1c1, which are responsible for sulfation of T4 in liver and kidneys, also did not significantly change in any group, except Sult1a1 in kidneys (Fig. 8). The mRNA expression of Sult1a1 in kidneys significantly increased in Tx rats compared with control rats (F(2,14) = 64.3, p b 0.01). In Tx rats infused with T3, the mRNA expression were significantly decreased compared with Tx rats (p b 0.01), and returned to control levels.

Discussion

Fig. 2. Serum [13C9]thyroxine (T4) concentrations after an intravenous administration of [13C9]T4 (1.5 μg/kg) to control and thyroidectomized (Tx) rats with infused vehicle or triiodothyronine (T3) (1.5 or 7.5 μg/head/day). [13C9]T4 was administered to all group rats on day 14 after subcutaneous implantation of vehicle or T3. All data represent the mean ± S.D. of five animals.

Hypothyroidism is the most common clinical disorder of thyroid function. It is caused by thyroid gland disorders including congenital hypothyroid disorders, previous thyroid surgery and irradiation, and pituitary and hypothalamic disease, which leads to a decrease in thyroidal production and secretion of T4 and T3. In general, when given in the proper dose, the treatment of choice for hypothyroidism is replacement of T4 which has a high degree of effectiveness and small risk of adverse reactions (Toft, 1994). T4 is metabolized to T3 in target tissues by deiodinases. T3 is the active form of thyroid hormone. Thus, T3 replacement therapy in hypothyroidism patients may be more effective than T4 replacement.

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Table 3 Hepatic and renal UGT and deiodinase type 1 activity of T4 in control and Tx rats. Tissue

Enzyme

Control

Liver

D1 UGT D1 UGT

1457.2 76.2 1802.6 60.4

Kidney

Tx ± ± ± ±

193.2 16.7 69.5 17.1

435.2 147.2 318.0 72.2

± ± ± ±

107.1** 16.0** 83.6** 19.6

Activity was expressed as pmol/min/g tissue. Data are expressed as the mean values ± S.D. (n = 6). **p b 0.01 compared with controls. D1, deiodinase type 1; UGT, uridine diphosphate-glucuronosyltransferase.

Tx rats are a well-established hypothyroidism model. We have reported that serum T4 and T3 levels are decreased, but not completely abolished, by surgical thyroidectomy. Further investigation indicated that T4 levels in Tx rats are maintained at a constant very low level by repression of T4 turnover in serum. In this study, we have investigated the mechanisms by which the serum T4 turnover is repressed after Tx, and the effects of T3 replacement on the expression and activity of deiodinases and other metabolic enzymes. Endogenous T4 and T3, and exogenous T4 ([13C9]T4) concentrations in serum in control and Tx rats were determined using an established on-line SPE LC-MS/MS method (Nagao et al., 2011). After Tx treatment, serum T4 and T3 levels significantly decreased, and the turnover rate of serum [13C9]T4 was decreased. T3 in Tx rats returned to physiological levels with T3 infusion. Moreover, after T3 infusion for 2 weeks, the T4 levels in Tx rats were reduced by approximately one-half compared with before T3 treatment. These results suggest that the repression of serum T4 disappearance after Tx treatment is a homeostatic response to decreased serum T3 concentrations. When the dose of T3 infusion was increased 5-fold, serum T3 levels of Tx rats increased, but by less than 2-fold. We speculate that this phenomenon is also a homeostatic response caused by accelerated T3 uptake into T3-deficient tissues. Some transporters involved in the uptake of thyroid hormones into cell include members of the organic anion transporting polypeptide (OATP) family (Hagenbuch, 2007) and the monocarboxylate transporter (MCT) family (Visser et al., 2007). These transporters may accelerate

T4 and T3 uptake into tissues in Tx rats after T3 infusion. However, further studies are required to clarify this mechanism. The turnover rate of serum [13C9]T4 returned to normal levels after infusion of T3. This suggests that the regulation of turnover rate of serum [13C9]T4 is a key factor for maintaining consistent serum T3 levels. T4 is mainly eliminated by metabolism via deiodinases, UGTs, and SULTs in several tissues. We speculated that decreased T4 metabolism by these enzymes is one mechanism by which serum T4 degradation is repressed in Tx rats. We determined activity and mRNA expression of D1, D2, UGT, and SULT in rats after Tx and T3 replacement. Hepatic and renal D1 activity was significantly decreased by Tx, but returned to normal levels with T3 infusion. These changes paralleled those of D1 mRNA levels in both tissues. These results strongly suggest that the expression of hepatic and renal D1 is regulated at the transcriptional level by serum T3 concentrations, and that this plays a major role in the suppression of serum T4 turnover in Tx rats. T3-induced increases in D1 activity and mRNA expression are demonstrated in rats, mice and humans (Berry et al., 1990, 1991). T3 exerts its actions on D1 gene expression by activating thyroid hormone receptor (TR) β. In humans, the functional TRβ complex consists of a heterodimer with retinoid X receptor (RXR) that binds to a thyroid hormone response element (TRE) located in the promoter region of D1 gene to modulate gene expression (Toyoda et al., 1995). However, the TRE in rats and mice has not yet been identified in the 5′-FR of the D1 promoter (Bianco et al., 2002; Maia et al., 1995a,b). Recently, it was reported that hepatocyte nuclear factor 4α (HNF4α), member of the nuclear receptor superfamily regulates the promoter of the D1 gene in mice, and would regulate cooperatively through transcriptional regulation of the mouse D1 gene with GATA4 and T3-induced Krüppel-like transcription factor 9 (KLF9) (Ohguchi et al., 2008). HNF4α is known to express highly in the liver, kidneys, and other tissues in mammals (Sladek and Seidel, 2001). In the present study, thus, it is indicated that change of D1 activity and mRNA expression by Tx may also regulate these mechanisms. D2 activity and mRNA levels in the brain, BAT, and skeletal muscles were markedly increased by Tx. Moreover, in Tx rats with T3 infusion, although D2 activity and mRNA in BAT decreased by approximately 50% compared with after Tx, brain and skeletal muscle activity and mRNA levels hardly changed. van Doorn et al. (1986) reported that

Fig. 3. Type 1 deiodinase activity and mRNA expression in liver and kidneys of control and thyroidectomized (Tx) rats with infused vehicle or triiodothyronine (T3) (1.5 μg/head/day). Activity in (A) liver and (B) kidneys. mRNA expression in (C) liver and (D) kidneys. Tissues were extracted day 14 after subcutaneous implantation of vehicle or T3. All data represent the mean ± S.D. of six animals. **p b 0.01 compared with the controls. #p b 0.05 and ##p b 0.01 compared with the Txs. The correlation coefficients of standard curves for RT-PCR were 1.0000 for D1 and gapdh in both tissues.

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Fig. 4. Type 2 deiodinase activity and mRNA expression in brain, brown adipose tissue (BAT) and skeletal muscles of control and thyroidectomized (Tx) rats with infused vehicle or triiodothyronine (T3) (1.5 μg/head/day). Activity in (A) brain, (B) BAT, and (C) skeletal muscles. mRNA expression in (D) brain, (E) BAT, and (F) skeletal muscles. Tissues were extracted on day 14 after subcutaneous implantation of vehicle or T3. All data represent the mean ± S.D. of six animals. *p b 0.05 and **p b 0.01 compared with the controls. ##p b 0.01 compared with the Txs. The correlation coefficients of D2 standard curves for RT-PCR were 0.9998 for brain, 0.9992 for BAT, and 0.9996 for skeletal muscles. The correlation coefficients of gapdh standard curves were higher than 0.9993 in all tissues.

hypothyroidism led to a significant loss of intracellular T3 in rat tissues (liver, cerebral cortex, thymus, testis, and BAT). Thus, our results suggest that increased D2 activity after Tx is a compensatory response to decreased intracellular T3. Intracellular T3 levels are known to be maintained by direct T3 uptake from blood and by intracellular T4 to T3 selective conversion by D2 (Bianco et al., 2002; Williams and Bassett, 2011). Furthermore, Bianco and Silva (1987), and van Doorn et al. (1986) reported that intracellular T3 concentrations in BAT and brain were supplied at a ratio of 50:50 and 20:80 from these two pathways, respectively. Our results strongly suggest that serum T3 levels in hypothyroid patients can be returned to physiological levels by T3 replacement, but that biological function is not fully restored in every tissue. Thus, these findings indicate the existence of tissue-specific mechanisms that regulate thyroid hormone concentrations in target tissues. Interestingly, hepatic T4-UGT activity was markedly increased by Tx, and returned to normal levels after T3 replacement. In contrast, there

was no significant difference in renal T4-UGT activity after Tx and T3 replacement. mRNA expression of Ugt1a1 and Ugt1a6, the UGT isoforms responsible for glucuronidation of T4 showed different responses in both tissues, and that the change in T4-UGT activity after Tx paralleled that in Ugt1a1 mRNA levels. Shelby et al. (2003) reported that the rat Ugt1a1 mRNA levels in liver and kidneys were approximately 3 to 5-fold higher than those of Ugt1a6. These findings suggest that UGT1A1 is the primary isoform participating in glucuronidation of T4 by UGTs in rat liver and kidney. We were surprised that this enzyme that increases serum T4 conversion into a biologically inactive form was actually increased in Tx rats. Glucuronidation is a so-called phase II detoxification reaction that increases the water solubility of the substrates and facilitates their biliary and urinary clearance. However, T4 glucuronide excreted in bile is deconjugated by bacterial β-glucuronidases in the intestines, and then some of the liberated T4 is reabsorbed, resulting in an enterohepatic circulation (Visser, 1996; Wu et al., 2005). Moreover, a recent study suggested that T4 glucuronide

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Fig. 7. (A) Hepatic and (B) renal T4-SULT activity in control and thyroidectomized (Tx) rats with infused vehicle or triiodothyronine (T3) (1.5 μg/head/day). SULT; sulfotransferase. Tissues were extracted on day 14 after subcutaneous implantation of vehicle or T3. All data represent the mean ± S.D. of six animals.

Fig. 5. (A) Hepatic and (B) renal T4-UGT activity in control and thyroidectomized (Tx) rats with infused vehicle or triiodothyronine (T3) (1.5 μg/head/day). UGT; uridine diphosphateglucuronosyltransferase. Tissues were extracted on day 14 after subcutaneous implantation of vehicle or T3. All data represent the mean ± S.D. of six animals. *p b 0.05 compared with the controls. #p b 0.05 compared with the Txs.

is stored in the kidney and contributes to maintaining serum T 4 homeostasis by being deconjugated to form T4 (Buitendijk and Galton, 2012). Thus, T4 glucuronide may serve as a storage form of T4. Our findings raise the possibility that T4-UGT activity in the liver

Fig. 6. Relative mRNA expression of Ugt1a isoforms in liver and kidneys of control and thyroidectomized (Tx) rats with infused vehicle or triiodothyronine (T3) (1.5 μg/head/day). (A) Ugt1a1 and (B) Ugt1a6 in liver. (C) Ugt1a1 and (D) Ugt1a6 in kidneys. UGT; uridine diphosphate-glucuronosyltransferase. Tissues were extracted on day 14 after subcutaneous implantation of vehicle or T3. All data represent the mean ± S.D. of six animals. *p b 0.05 and **p b 0.01 compared with the controls. #p b 0.05 and ##p b 0.01 compared with the Txs. The correlation coefficients of Ugt1a1 and Ugt1a6 standard curves for RT-PCR were 1.0000 and 0.9999 for liver, and 0.9998 and 1.0000 for kidneys, respectively.

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Fig. 8. Relative mRNA expression of Sult1 isoforms in liver and kidneys of control and thyroidectomized (Tx) rats with infused vehicle or triiodothyronine (T3) (1.5 μg/head/day). (A) Sult1a1 and (B) Sult1c1 in liver. (C) Sult1a1 and (D) Sult1c1 in kidneys. SULT; sulfotransferase. Tissues were extracted on day 14 after subcutaneous implantation of vehicle or T3. All data represent the mean ± S.D. of six animals. **p b 0.01 compared with the controls. ##p b 0.01 compared with the Txs. The correlation coefficients of Sult1a1 and Sult1c1 standard curves for RT-PCR were 1.0000 and 0.9960 for liver, and 0.9998 and 1.0000 for kidneys, respectively.

increases in order to indirectly maintain the active form T3 that is decreased by Tx, and that T4 glucuronide production is thus accelerated. Estrogens are known to be regulated by a similar mechanism. Estrone sulfate is stored in the body, but is deconjugated to the pre-active form estrone, and then reduced to the active form, estradiol (Purohit et al., 2011). There were no significant differences in hepatic and renal T4-SULT activity in Tx rats compared with control rats or T3-treated rats. Additionally, hepatic Sult1a1 mRNA levels also did not change. These results are in agreement with a previous report by Dunn and Klaassen (2000). In rats, SULT1 family members are expressed at the highest levels in the liver compared with other tissues (Dunn and Klaassen, 1998). Hepatic SULT1A1 is regulated by GA-binding protein (GABP) of the EST transcription factor family (Hempel et al., 2004). RodriguezPena et al. (2002) reported that GABP levels in rat liver were not changed by Tx. These findings are consistent with our results. Furthermore, hepatic SULT1A1 and SULT1C1 are known to be regulated by glucocorticoids and growth hormone, respectively (Runge-Morris and Kocarek, 2005; Liu and Klaassen, 1996). These findings suggest that SULTs are not involved in the suppression of serum T4 turnover in Tx rats.

Conclusion In this study, we report that repression of the disappearance of serum T 4 in rats after Tx treatment is a homeostatic response to decreased serum T3 . Repression of the deiodination of T 4 by D1 in the liver and kidneys may be a primary mechanism of the homeostatic response. Interestingly, increased UGT activity in the liver may also be involved in this homeostatic response. Namely, T 4 glucuronide may be a storage form of thyroid hormones . Moreover, it is indicated the possibility that T 3 replacement therapy could not completely normalize thyroid hormone homeostasis in hypothyroidism patients.

Conflict of interest statement The authors declare no conflict of interest.

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