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Effect of T3 Treatment and Food Ration on Hepatic Deiodination and Conjugation of Thyroid Hormones in Rainbow Trout, Oncorhynchus mykiss
General and Comparative Endocrinology 115, 379–386 (1999) Article ID gcen.1999.7325, available online at http://www.idealibrary.com on
Effect of T3 Treatment and Food Ration on Hepatic Deiodination and Conjugation of Thyroid Hormones in Rainbow Trout, Oncorhynchus mykiss K. W. Finnson and J. G. Eales Department of Zoology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada Accepted May 21, 1999
We studied the 7-day effects of 3,5,38-triiodothyronine (T3) hyperthyroidism (induced by 12 ppm T3 in food) and food ration (0, 0.5, or 2% body weight/day) on in vitro hepatic glucuronidation, sulfation, and deiodination of thyroxine (T4), T3, and 3,38,58-triiodothyronine (rT3). T3 treatment doubled plasma T3 with no change in plasma T4, depressed hepatic low-Km (1 nM) outer-ring deiodination (ORD) of T4, induced low-Km (1 nM) inner-ring deiodination (IRD) of both T4 and T3 but did not alter high-Km (1 M) rT3ORD, glucuronidation, or sulfation of T4, T3, or rT3. Plasma T4 levels were greater for 0 and 2% rations than for a 0.5% ration. Fasting decreased low-Km T4ORD activity and increased high-Km rT3ORD activity but did not alter T4IRD or T3IRD activities. T4, T3, and rT3 glucuronidation were greater for 0 and 0.5% rations than for a 2% ration. T3 glucuronidation was greater for a 0.5% ration than for a 0% ration. T3 and rT3 sulfation were greater for a 2% ration than for a 0 or a 0.5% ration; ration did not change T4 sulfation. We conclude that (i) modest experimental T3 hyperthyroidism induces T3 autoregulation by adjusting hepatic low-Km ORD and IRD activities but not high-Km rT3ORD or conjugation activities; (ii) in contrast, ration level changes both deiodination and conjugation pathways, suggesting that the response to ration does not solely reflect altered T3 production; (iii) deiodination and conjugation appear complementary in regulating thyroidal status in response to ration; and (iv) high-Km rT3ORD in trout differs from rat type I deiodination in that it does not respond to T3 hyperthyroidism and it increases, rather than decreases, its activity during fasting. r 1999 Academic Press
0016-6480/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
INTRODUCTION Thyroxine (T4 ), the main thyroid hormone (TH) released from the fish thyroid gland (reviewed by Eales and Brown, 1993), is considered a prohormone because of its modest intrinsic activity. It becomes activated when converted to 3,5,38-triiodothyronine (T3 ) by outer-ring deiodination (ORD) in peripheral tissues. Both T4 and T3 undergo inner-ring deiodination (IRD), producing inactive 3,38,58-triiodothyronine (reverse T3, rT3 ) and 3,38-diiodothyronine (T2 ), respectively. These TH ORD and IRD conversions are catalyzed by microsomal deiodinases which function at low T4 substrate concentrations (Km ⫽ 1 nM) in liver and other tissues. They represent key pathways for regulating T3 availability and hence fish thyroidal status (Eales and Brown, 1993). Recently, rT3ORD activity, which functions in the high nanomolar range (high-Km ), has been reported for trout liver (Orozco et al., 1997; Finnson et al., 1999) and tilapia kidney (Mol et al., 1993, 1997). Factors regulating high-Km rT3ORD activity have not been studied. Conjugation is an alternate major pathway for TH metabolism. It involves linking either glucuronic acid or sulfate to the TH 48-hydroxyl group (Visser, 1994). These reactions are catalyzed, respectively, by glucuronosyltransferases and sulfotransferases, which have been characterized in liver of rainbow trout, Oncorhynchus mykiss (Finnson and Eales, 1997, 1998). Both glucuronidated and sulfated TH are produced by 379
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isolated hepatocytes and occur in bile of trout injected with [ 125I]T4 or T3 (Finnson and Eales, 1996). Over 40% of the injected label may be present in trout bile 24 h post-injection, with most radioactivity occurring as conjugates (Eales and Brown, 1993). TH-glucuronides also occur in significant levels in tilapia plasma (DiStefano et al., 1998). These observations indicate the potential of both glucuronidation and sulfation pathways to regulate TH homeostasis and thyroidal status in fish. If conjugation pathways contribute to regulation of fish thyroidal status, then experimental change in thyroidal status should alter the activities of enzymes involved in TH conjugation. To investigate this possibility we measured rates of hepatic glucuronidation and sulfation of T4, T3, and rT3 in trout in which thyroidal status was altered in two ways. First, we fed T3-supplemented food to create a T3 challenge (Eales and Finnson, 1991; Sweeting and Eales, 1992b). Second, we varied food ration, which indirectly changes thyroidal status (Sweeting and Eales, 1992c). We also measured TH deiodination to investigate any relationships between deiodination and conjugation pathways, since it has been shown that deiodination and conjugation are interrelated pathways for TH metabolism in mammals (Visser et al., 1990; Visser, 1994).
MATERIALS AND METHODS Fish Maintenance Rainbow trout were obtained from the Rockwood Hatchery, Balmoral, Manitoba and held in the laboratory in flowing, aerated, dechlorinated water at 12°C, on a 12L:12D photoperiod. They were fed a commercial diet (Martin Feed Mills, Elmira, Ontario; trout feed pellets, 3.2 mm diameter) once daily at a 1.0% body weight (BW) ration for 4 days prior to start of treatment.
T3 Treatment Food was prepared by spraying trout pellets with either ethanol (control) or ethanol containing T3 (nominal concentration in food ⫽ 12 ppm) (experimental) and dried under a fumehood. Groups of six to seven fish were assigned to four tanks. Two control tanks were fed 1.0% body weight/day (BW/d) with control
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Finnson and Eales
pellets and two experimental tanks were fed 1.0% BW/d with T3-treated pellets. All trout were fed for 7 days.
Food Ration Groups of six to seven fish were assigned to six tanks. Fish in two tanks were fasted (0% BW/d), while fish in two tanks were fed 0.5% BW/d and fish in two other tanks were fed 2.0% BW/d for 7 days.
Blood Sampling and Subcellular Fractions Trout were anesthetized in tricaine methanesulfonate (0.07 g/L) and weighed and blood samples were removed from caudal vessels using a heparinized syringe. Plasma was separated by centrifugation and stored at ⫺76°C. Trout were then killed by concussion; livers were removed and rinsed with ice-cold 0.1 M Tris–HCl buffer (pH 7.2) containing 0.25 M sucrose, 1 mM EDTA, and 10 mM ditheothrietrol (DTT). Livers were weighed to determine hepatosomatic index (HSI ⫽ liver weight ⫻ 100/body weight) and each liver was added to 4 vol (w/v) of buffer, minced with scissors, and dispensed into a 55-ml Wheaton homogenization chamber. Tissue was homogenized by two strokes of a motorized pestle (Tri-R Instruments, Inc., NY). This initial homogenate was filtered through two layers of cheesecloth and transferred to plastic centrifuge tubes. The resulting homogenate was centrifuged for 20 min at 730g to obtain the crude nuclear pellet. The post-nuclear supernatant was centrifuged for 20 min at 25,200g to obtain the crude mitochondrial/ lysosomal pellet. The post-mitochondrial/lysosomal supernatant was centrifuged for 67 min at 110,000g to separate the microsomal pellet from the cytosol. A 1-ml aliquot of cytosolic fraction was obtained and microsomal pellets were resuspended in 1–2 ml of buffer. Cytosolic and microsomal fractions were stored at ⫺76°C (Shields and Eales, 1986).
Radioimmunoassay (RIA) Plasma T4 and T3 levels were measured simultaneously, using a solid-phase RIA (Omeljaniuk et al., 1984).
Glucuronosyltransferase (GT) Assay Microsomal fractions were thawed on ice and diluted (⬃1:25) with 0.1 M Tris–HCl buffer (pH 7.8). A
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volume of 500 µl of diluted microsomal fraction was added to siliconized test tubes containing 20 µl of the cofactor uridine 58-diphosphoglucuronic acid (UDPGA, Sigma) previously dissolved in buffer (nominal UDPGA conc. ⫽ 0.25 mM). Control tubes received 20 µl of buffer without UDPGA. All tubes were vortexed and equilibrated in darkness for 15 min in a water bath (12°C; 140 rpm). The reaction was started by adding 20 µl of either 125I-labeled T4, T3, or rT3 (New England Nuclear) previously dissolved in 0.1 N NaOH (final conc. ⫽ 1 µM; ⬃100,000 cpm). The reaction was stopped after 60 min by adding 1 ml of chloroform/methanol (2:1 v/v). This mixture was vortexed, centrifuged for 5–10 min at 1420g and a 250-µl aliquot of the aqueous methanol layer was added to Sephadex LH-20 minicolumns containing 750 µl of 1.0 N HCl. Columns were swirled and drained and 3 ml of 0.1 N HCl was added, which was eluted to waste (iodide fraction). TH glucuronide fraction was then eluted with 8 ml of H2O and counted in a well gamma detector (Finnson and Eales, 1996). TH glucuronide formation was determined by subtracting cpm of control tubes (⫺UDPGA) from cpm of experimental tubes (⫹UDPGA) and GT activity was expressed as pmol TH glucuronidated/mg protein/h (Finnson and Eales, 1997).
Sulfotransferase (ST) Assay Cytosolic fractions were thawed on ice and diluted (⬃1:25) with 0.1 M Tris–HCl buffer (pH 7.0). A volume of 500 µl of diluted cytosolic fraction was added to siliconized test tubes containing 20 µl of the cofactor 38-phosphoadenosine 58-phosphosulfate (PAPS, Sigma) previously dissolved in buffer (nominal PAPS conc. ⫽ 20 µM). Control tubes received 20 µl of buffer without PAPS. Incubation and extraction procedures were identical to those for the GT assay and the resulting aqueous methanol layer was added to Sephadex LH-20 minicolumns containing 750 µl of 1.0 N HCl. Columns were swirled and drained and 3 ml of 0.1 N HCl was added, which was eluted to waste (iodide). The TH sulfate fraction was then eluted with 8 ml of water and counted in a well gamma detector (Finnson and Eales, 1996). TH sulfate formation was determined by subtracting cpm from control tubes (⫺PAPS) from cpm of experimental tubes (⫹PAPS) and ST activity was expressed as pmol TH sulfated/mg protein/h (Finnson and Eales, 1998).
Deiodination Assay This was based on the method of Shields and Eales (1986). Microsomal fractions were thawed on ice and diluted (⬃1:25) with 0.1 M Tris–HCl buffer (pH ⫽ 7.2) containing 10 mM of DTT and 1 mM of EDTA to achieve a final protein concentration of 0.2–0.8 mg/ml. A volume of 500 µl of diluted microsomal fraction was added to siliconized test tubes and equilibrated in darkness in a water bath (12°C; 140 rpm). The reaction was started by adding 100,000 cpm of either 125Ilabeled T4 or T3 (final conc. ⫽ 1 nM) or rT3 (final conc. ⫽ 1 µM) previously dissolved in 0.1 N NaOH. The reaction was stopped after 60 min by adding 1 ml of chloroform/methanol (2:1 v/v). This mixture was vortexed and centrifuged for 5–10 min at 1420g and a 200-µl aliquot of the aqueous methanol layer was used for HPLC analysis. Deiodination (ORD or IRD) activity was expressed as pmol TH (T4, T3, or rT3 ) deiodinated/mg protein/h.
HPLC Analysis HPLC analyses of deiodination products were conducted with a Gilson IBM binary gradient system using two solvent systems: (A) acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA)/H2O containing 0.1% TFA, ACN gradient increasing linearly from 42 to 54% (10–20 min); and (B) ACN/0.02 M ammonium acetate (pH 4.0), ACN increasing linearly from 47 to 60% (0–25 min) (Sweeting and Eales, 1992a).
Statistics Statistical analyses were performed using either Student’s t test or one-way analysis of variance (ANOVA) followed by Student–Neuman–Kuels test. Levine test was used to confirm homogeneity of variance. Treatments were considered different if P ⬍ 0.05.
RESULTS T3 Treatment T3 treatment decreased HSI (Table 1) and elevated plasma T3 levels but did not alter plasma T4 levels (Fig.
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TABLE 1 Effect of T3 Treatment (Fed a 1% Ration Containing 12 ppm T3 ) and Food Ration (0, 0.5, and 2.0% BW) on Mean (⫾SEM) Body Weight (BW) and Hepatosomatic Index (HSI) of Rainbow Trout Study
Treatment
Weight (g)
HSI (%)
1
Control T3 (12 ppm) Ration 0% Ration 0.5% Ration 2.0%
294 ⫾ 18.7 279 ⫾ 14.3 170 ⫾ 22.6 194 ⫾ 22.9 256 ⫾ 14.7 b
1.4 ⫾ 0.1 1.2 ⫾ 0.1 a 0.7 ⫾ 0.1 0.9 ⫾ 0.1 1.4 ⫾ 0.1 b
2
a HSI value in T -treated trout was significantly lower than in 3 control trout (P ⬍ 0.05). b Body weight and HSI were significantly higher in trout fed a 2.0% BW ration than in trout fed lower (0 and 0.5% BW) rations (P ⬍ 0.05).
1). T3 treatment also decreased hepatic T4ORD activity, increased T4IRD and T3IRD activities but did not change rT3ORD activity (Fig. 2). T3ORD and rT3IRD activities were negligible (data not shown). T3 treatment had no effect on glucuronidation (Fig. 3) or sulfation (Fig. 4) of T4, T3, or rT3.
FIG. 2. Mean (⫾SEM) hepatic T4ORD, T4IRD, T3IRD, and rT3ORD activities of control and T3-treated trout. T4 substrate, 1 nM; T3 substrate, 1 µM. aDifferent from control (P ⬍ 0.05). No T3IRD or rT3IRD activities were detected.
Food Ration Both body weight and HSI were higher in trout fed a 2.0% ration than in trout fed lower (0 and 0.5%) rations
FIG. 1. Mean (⫾SEM) plasma T4 and T3 concentrations of control and T3-treated trout. aHigher than control (P ⬍ 0.05).
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FIG. 3. Mean (⫾SEM) hepatic glucuronidation of T4, T3, and rT3 of control or T3-treated trout. There were no significant differences between treatments (P ⬍ 0.05).
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FIG. 4. Mean (⫾SEM) hepatic sulfation of T4, T3, and rT3 of control and T3-treated trout. There were no significant differences between treatments (P ⬍ 0.05).
(Table 1). Plasma T4 levels were lower in trout fed a 0.5% ration than in trout fed either a 0 or a 2.0% ration but ration had no effect on plasma T3 concentration (Fig. 5). Fasting decreased T4ORD activity and increased rT3ORD activity relative to 0.5 and 2.0%
FIG. 6. Mean (⫾SEM) hepatic T4ORD and rT3ORD activity of trout fed rations of 0, 0.5, or 2.0% BW for 7 days. T4 substrate, 1 nM; rT3 substrate, 1 µM. aSignificantly different 0.5 and 2% (P ⬍ 0.05). No T4IRD, T3ORD, T3IRD, or rT3IRD activities were detected.
rations (Fig. 6). T4IRD, T3ORD, T3IRD, or rT3IRD activities were negligible in trout fed any ration (data not shown). Glucuronidation of T4, T3, and rT3 was greater in trout fed 0 or 0.5% rations than in trout fed 2.0%, and glucuronidation of T3 was greater in starved trout than in trout fed 0.5% (Fig. 7). Sulfation of both T3 and rT3 was greater in trout fed a 2.0% ration than in trout fed 0 and 0.5% rations, ration did not effect T4 sulfation (Fig. 8).
DISCUSSION
FIG. 5. Mean (⫾SEM) plasma T4 and T3 concentrations of trout fed rations 0, 0.5, or 2.0% BW for 7 days. aSignificantly lower than 0 or 2.0% BW (P ⬍ 0.05).
Both hyperthyroidism (T3 treatment) and food ration alter thyroidal status in trout by acting on deiodination pathways (reviewed by Eales and Brown, 1993). Our present goal was to determine if these conditions also influence hepatic TH conjugation pathways, and at the same time we also studied TH deiodination. This allows a comparison to mammalian deiodination and conjugation pathways which are shown to interact (Visser et al., 1990; Visser, 1994). Effects of T3 treatment and food ration on trout hepatic TH conjugation and deiodination are summarized in Table 2.
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FIG. 7. Mean (⫾SEM) hepatic glucuronidation of T4, T3, and rT3 of trout fed rations of 0, 0.5, or 2.0% BW for 7 days. aSignificantly lower for 2% than for lower rations (P ⬍ 0.05). bSignificantly lower than for fasted trout (P ⬍ 0.05).
The present responses of trout to experimental T3 hyperthyroidism confirm previous studies in showing reduced hepatic T4ORD activity (< T3 production) and increased IRD activity for both T4 (> rT3 prodution) and T3 (> T2 production) (Eales and Finnson, 1991; Sweeting and Eales, 1992c; MacLatchy and Eales, 1992). All the above changes in hepatic deiodination would contribute to T3 autoregulation. Our novel findings are the negligible effects of T3 hyperthyroidism on glucuronidation or sulfation of T4 , T3 , or rT3 , which also have the potential to contribute to T3 autoregulation. Furthermore, we found no change in high-Km rT3 deiodination, even though formation of rT3 is induced by T3 treatment. Therefore, at least under our experimental conditions, autoregulation of T3 involved modifying only the hepatic low-Km T4ORD, T4IRD, and T3IRD activities. The responses of the trout thyroid system to changes in food ration or fasting confirm previous studies in showing reduced hepatic T4ORD activity following a 7-day fast (Sweeting and Eales, 1992c). The effects of food ration or fasting on trout hepatic T4IRD and T3IRD activities have not been studied previously. Our data show that these inner-ring deiodination pathways are insensitive to food intake. However, fasting did
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Finnson and Eales
FIG. 8. Mean (⫾SEM) hepatic sulfation of T4, T3, and rT3 of trout fed rations of 0, 0.5, or 2.0% BW for 7 days. aSignificantly higher for 2% than for lower rations (P ⬍ 0.05).
increase the hepatic high-Km rT3ORD activity in trout. This suggests a greater need to degrade rT3 during a fast, despite no induction of hepatic rT3 formation. Regardless of the physiological implications, the fasting response of high-Km rT3ORD in the trout is the opposite to that observed in mammals due to type I deiodination (McNabb, 1992). For all TH tested, the highest food ration (2% BW/d) decreased hepatic TH glucuronidation. Glucuronidation is a major pathway for biliary excretion of TH in
TABLE 2 Summary of the Significant (P ⬍ 0.05) Effects of T3 Treatment and Fasting on Outer- and Inner-Ring Deiodination (ORD and IRD), Glucuronidation (Gluc), and Sulfation (Sulf) of TH in Liver of Rainbow Trout Treatment
Pathway
T4
T3
T3-treated
ORD IRD Gluc Sulf ORD IRD Gluc Sulf
⫺ ⫹
⫹
Fasted
⫺ ⫹
rT3
⫹ ⫹ ⫺
⫹ ⫺
Note. Positive (⫹) sign indicates stimulation and negative (⫺) sign indicates inhibition.
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trout (Finnson and Eales, 1996) and so a high food intake would likely decrease biliary TH loss. Reduced biliary loss of T4 and T3 should increase availability of T3 and its T4 precursor. This, coupled with increased hepatic T4ORD activity, should increase T3 availability. Therefore, both depressed TH glucuronidation and increased T4ORD may be complementary in enhancing T3 availability and thyroidal status when food intake is high. This is consistent with the general effect of food intake to elevate thyroidal status in fish. The highest food ration (2% BW/d) increased hepatic sulfation of T3 and rT3 in trout. This is the response opposite to that described above for glucuronide conjugates. Since THS occur in trout bile (Finnson and Eales, 1996), this suggests that the high food ration favors excretion of TH and particularly rT3 as sulfate conjugates. However, the biological significance of sulfation may vary with the type of TH. In contrast to T4S and rT3S, T3S can be desulfated more readily in trout by hepatic mitochondrial/lysosomal and microsomal cell fractions and by isolated hepatocytes to regenerate T3 (Finnson and Eales, 1999). Thus, as suggested for mammals, T3S may act as a temporary inactive lipid-insoluble storage form of T3 that can be reactivated by desulfation (Kung et al., 1988; Santini et al., 1992). Therefore, the enhanced T3 sulfation accompanying the high ration could complement changes in deiodination and glucuronidation to enhance thyroidal status by favoring sequestration of T3 in liver cells. Although both T3 treatment and food intake influence thyroid function, their effects on hepatic TH deiodination and conjugation pathways differ. Therefore the thyroid response to food involves adjustments that do not depend solely on changes in T3 production. There are few studies in other vertebrates on the effects of thyroid status, nutritional status, or other physiological states on TH conjugation. However, in general agreement with our study, Kaptein et al. (1997) showed that rat hepatic sulfation of TH was decreased by fasting but was unaffected by experimental hyperthyroidism (T4 treatment). Gong et al. (1992) showed that rat hepatic sulfation of T3 was regulated by the pattern of pituitary growth hormone secretion. It has also been shown that formation and metabolism of TH conjugation may depend on the stage of development. TH sulfates occur in plasma of fetal lambs (Visser, 1994); Wu et al. (1998) found sulfate conjugates of rT3 and
3,38-T2 in plasma of premetamorphic tadpoles. Further studies on sulfation of TH in different species are needed to assess the role of sulfation in regulating thyroidal status. Under our in vitro assay conditions, both glucuronidation and sulfation showed a substrate preference of rT3 ⬎ T4 ⫽ T3 (Figs. 3 and 4). This is consistent with previous studies on trout (Finnson and Eales, 1997, 1998). Under normal conditions hepatic rT3 production is negligible in trout (Sweeting and Eales, 1992b; Johnston et al., 1996) and rT3 conjugation may therefore be of little physiological consequence. However, under conditions of TH challenge, T4 and T3IRD pathways are induced (Fig. 2) and preferential conjugation and removal of rT3 may then be important. Trout hepatic high-Km rT3ORD activity was unaffected by a T3 challenge but increased during fasting. In both regards the trout high-Km rT3ORD activity differs from rat type I deiodination activity, which increases with hyperthyroidism and decreases during fasting (McNabb, 1992; Kohrle, 1996). Clearly there are major functional differences between trout high-Km rT3ORD and rat type I deiodination. Other differences between these two enzyme systems are summarized by Finnson et al. (1999). We conclude that hepatic TH conjugation pathways are insensitive to experimental T3 hyperthyroidism but do respond to food ration. Both glucuronide and sulfate conjugation may in different ways complement hepatic deiodination in regulating T3 availability and TH homeostasis of trout in different nutritional states. Finally, the high-Km rT3ORD differs from rat type I deiodination in its response to both T3 hyperthyroidism and nutritional state.
ACKNOWLEDGMENTS This study was supported by Natural Sciences and Research Council of Canada Research Grant A1965 to J. G. E.
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Finnson and Eales Mol, K. A., Van Der Geyton, S., Darras, V. M., Visser, T. J., and Kuhn, E. R. (1997). Characterization of iodothyronine outer ring and inner ring deiodinase activities in the blue tilapia, Oreochromis aureus. Endocrinology 138, 1787–1793. Mol, K. A., Kaptein, E., Darras, V. M., deGreef, W. J., Kuhn, E. R., and Visser, T. J. (1993). Different thyroid hormone-deiodinating enzymes in tilapia (Oreochromis niloticus) liver and kidney. FEBS Lett. 321, 140–144. Omeljaniuk, R. J., Cook, R. F., and Eales, J. G. (1984). Simultaneous measurement of thyroxine and triiodothyronine in trout plasma using a solid-phase radioimmunoassay. Can. J. Zool. 62, 1450–1453. Orozco, A., Silva, J. E., and Valverde, C. R. (1997). Rainbow trout expresses two iodothyronine phenolic ring deiodinase pathways with the characteristics of types I and II 58-deiodinases. Endocrinology 138, 254–258. Santini, F., Chopra, I. J., Wu, S.-Y., Solomon, D. H., and Teco, G. N. C. (1992). Metabolism of 3,5,38-triiodothyronine sulfate by tissues of the fetal rat: A consideration of the role of desulfation of 3,5,38triiodothyronine sulfate as a source of T3. Pediatr. Res. 3, 541–544. Shields, C. A., and Eales, J. G. (1986). Thyroxine 58-monodeiodinase activity in hepatocytes of rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 63, 334–343. Sweeting, R. M., and Eales, J. G. (1992a). HPLC analysis of in vitro deiodination products of thyroid hormones in the rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 85, 367–375. Sweeting, R. M., and Eales, J. G. (1992b). The acute influence of ingested thyroid hormones on hepatic deiodination pathways in the rainbow trout, Oncorhynchus mykiss. Can. J. Zool. 70, 1516–1525. Sweeting, R. M., and Eales, J. G. (1992c). The effects of fasting and feeding on hepatic thyroxine 58-monodeiodinase activity in the rainbow trout, Oncorhynchus mykiss. Can. J. Zool. 70, 1516–1525. Visser, T. J. (1994). Role of sulfation in thyroid hormone metabolism. Chemico-Biological Interactions 92, 293–303. Visser, T. J., van Buuren, C. J. C., Rutgers, M., Eelkman Rooda, S. J., and de Herder, W. W. (1990). The role of sulfation in thyroid hormone metabolism. Trends Endocrinol. Metab. 3, 211–218. Wu, S. Y., Huang, W. S., Polk, D. H., Parker, L. N., Fisher, D. A., and Galton, V. A. (1998). ‘‘Sulfoconjugation Is a Major Pathway of Thyroid Hormone Metabolism in Developing Animals: From Tadpoles to Mammals,’’ Proc. of the 80th Annual Meet. of The Endocrine Society, 1998. New Orleans.
Effect of T3 Treatment and Food Ration on Hepatic Deiodination and Conjugation of Thyroid Hormones in Rainbow Trout, Oncorhynchus mykiss K. W. Finnson and J. G. Eales Department of Zoology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada Accepted May 21, 1999
We studied the 7-day effects of 3,5,38-triiodothyronine (T3) hyperthyroidism (induced by 12 ppm T3 in food) and food ration (0, 0.5, or 2% body weight/day) on in vitro hepatic glucuronidation, sulfation, and deiodination of thyroxine (T4), T3, and 3,38,58-triiodothyronine (rT3). T3 treatment doubled plasma T3 with no change in plasma T4, depressed hepatic low-Km (1 nM) outer-ring deiodination (ORD) of T4, induced low-Km (1 nM) inner-ring deiodination (IRD) of both T4 and T3 but did not alter high-Km (1 M) rT3ORD, glucuronidation, or sulfation of T4, T3, or rT3. Plasma T4 levels were greater for 0 and 2% rations than for a 0.5% ration. Fasting decreased low-Km T4ORD activity and increased high-Km rT3ORD activity but did not alter T4IRD or T3IRD activities. T4, T3, and rT3 glucuronidation were greater for 0 and 0.5% rations than for a 2% ration. T3 glucuronidation was greater for a 0.5% ration than for a 0% ration. T3 and rT3 sulfation were greater for a 2% ration than for a 0 or a 0.5% ration; ration did not change T4 sulfation. We conclude that (i) modest experimental T3 hyperthyroidism induces T3 autoregulation by adjusting hepatic low-Km ORD and IRD activities but not high-Km rT3ORD or conjugation activities; (ii) in contrast, ration level changes both deiodination and conjugation pathways, suggesting that the response to ration does not solely reflect altered T3 production; (iii) deiodination and conjugation appear complementary in regulating thyroidal status in response to ration; and (iv) high-Km rT3ORD in trout differs from rat type I deiodination in that it does not respond to T3 hyperthyroidism and it increases, rather than decreases, its activity during fasting. r 1999 Academic Press
0016-6480/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
INTRODUCTION Thyroxine (T4 ), the main thyroid hormone (TH) released from the fish thyroid gland (reviewed by Eales and Brown, 1993), is considered a prohormone because of its modest intrinsic activity. It becomes activated when converted to 3,5,38-triiodothyronine (T3 ) by outer-ring deiodination (ORD) in peripheral tissues. Both T4 and T3 undergo inner-ring deiodination (IRD), producing inactive 3,38,58-triiodothyronine (reverse T3, rT3 ) and 3,38-diiodothyronine (T2 ), respectively. These TH ORD and IRD conversions are catalyzed by microsomal deiodinases which function at low T4 substrate concentrations (Km ⫽ 1 nM) in liver and other tissues. They represent key pathways for regulating T3 availability and hence fish thyroidal status (Eales and Brown, 1993). Recently, rT3ORD activity, which functions in the high nanomolar range (high-Km ), has been reported for trout liver (Orozco et al., 1997; Finnson et al., 1999) and tilapia kidney (Mol et al., 1993, 1997). Factors regulating high-Km rT3ORD activity have not been studied. Conjugation is an alternate major pathway for TH metabolism. It involves linking either glucuronic acid or sulfate to the TH 48-hydroxyl group (Visser, 1994). These reactions are catalyzed, respectively, by glucuronosyltransferases and sulfotransferases, which have been characterized in liver of rainbow trout, Oncorhynchus mykiss (Finnson and Eales, 1997, 1998). Both glucuronidated and sulfated TH are produced by 379
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isolated hepatocytes and occur in bile of trout injected with [ 125I]T4 or T3 (Finnson and Eales, 1996). Over 40% of the injected label may be present in trout bile 24 h post-injection, with most radioactivity occurring as conjugates (Eales and Brown, 1993). TH-glucuronides also occur in significant levels in tilapia plasma (DiStefano et al., 1998). These observations indicate the potential of both glucuronidation and sulfation pathways to regulate TH homeostasis and thyroidal status in fish. If conjugation pathways contribute to regulation of fish thyroidal status, then experimental change in thyroidal status should alter the activities of enzymes involved in TH conjugation. To investigate this possibility we measured rates of hepatic glucuronidation and sulfation of T4, T3, and rT3 in trout in which thyroidal status was altered in two ways. First, we fed T3-supplemented food to create a T3 challenge (Eales and Finnson, 1991; Sweeting and Eales, 1992b). Second, we varied food ration, which indirectly changes thyroidal status (Sweeting and Eales, 1992c). We also measured TH deiodination to investigate any relationships between deiodination and conjugation pathways, since it has been shown that deiodination and conjugation are interrelated pathways for TH metabolism in mammals (Visser et al., 1990; Visser, 1994).
MATERIALS AND METHODS Fish Maintenance Rainbow trout were obtained from the Rockwood Hatchery, Balmoral, Manitoba and held in the laboratory in flowing, aerated, dechlorinated water at 12°C, on a 12L:12D photoperiod. They were fed a commercial diet (Martin Feed Mills, Elmira, Ontario; trout feed pellets, 3.2 mm diameter) once daily at a 1.0% body weight (BW) ration for 4 days prior to start of treatment.
T3 Treatment Food was prepared by spraying trout pellets with either ethanol (control) or ethanol containing T3 (nominal concentration in food ⫽ 12 ppm) (experimental) and dried under a fumehood. Groups of six to seven fish were assigned to four tanks. Two control tanks were fed 1.0% body weight/day (BW/d) with control
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Finnson and Eales
pellets and two experimental tanks were fed 1.0% BW/d with T3-treated pellets. All trout were fed for 7 days.
Food Ration Groups of six to seven fish were assigned to six tanks. Fish in two tanks were fasted (0% BW/d), while fish in two tanks were fed 0.5% BW/d and fish in two other tanks were fed 2.0% BW/d for 7 days.
Blood Sampling and Subcellular Fractions Trout were anesthetized in tricaine methanesulfonate (0.07 g/L) and weighed and blood samples were removed from caudal vessels using a heparinized syringe. Plasma was separated by centrifugation and stored at ⫺76°C. Trout were then killed by concussion; livers were removed and rinsed with ice-cold 0.1 M Tris–HCl buffer (pH 7.2) containing 0.25 M sucrose, 1 mM EDTA, and 10 mM ditheothrietrol (DTT). Livers were weighed to determine hepatosomatic index (HSI ⫽ liver weight ⫻ 100/body weight) and each liver was added to 4 vol (w/v) of buffer, minced with scissors, and dispensed into a 55-ml Wheaton homogenization chamber. Tissue was homogenized by two strokes of a motorized pestle (Tri-R Instruments, Inc., NY). This initial homogenate was filtered through two layers of cheesecloth and transferred to plastic centrifuge tubes. The resulting homogenate was centrifuged for 20 min at 730g to obtain the crude nuclear pellet. The post-nuclear supernatant was centrifuged for 20 min at 25,200g to obtain the crude mitochondrial/ lysosomal pellet. The post-mitochondrial/lysosomal supernatant was centrifuged for 67 min at 110,000g to separate the microsomal pellet from the cytosol. A 1-ml aliquot of cytosolic fraction was obtained and microsomal pellets were resuspended in 1–2 ml of buffer. Cytosolic and microsomal fractions were stored at ⫺76°C (Shields and Eales, 1986).
Radioimmunoassay (RIA) Plasma T4 and T3 levels were measured simultaneously, using a solid-phase RIA (Omeljaniuk et al., 1984).
Glucuronosyltransferase (GT) Assay Microsomal fractions were thawed on ice and diluted (⬃1:25) with 0.1 M Tris–HCl buffer (pH 7.8). A
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volume of 500 µl of diluted microsomal fraction was added to siliconized test tubes containing 20 µl of the cofactor uridine 58-diphosphoglucuronic acid (UDPGA, Sigma) previously dissolved in buffer (nominal UDPGA conc. ⫽ 0.25 mM). Control tubes received 20 µl of buffer without UDPGA. All tubes were vortexed and equilibrated in darkness for 15 min in a water bath (12°C; 140 rpm). The reaction was started by adding 20 µl of either 125I-labeled T4, T3, or rT3 (New England Nuclear) previously dissolved in 0.1 N NaOH (final conc. ⫽ 1 µM; ⬃100,000 cpm). The reaction was stopped after 60 min by adding 1 ml of chloroform/methanol (2:1 v/v). This mixture was vortexed, centrifuged for 5–10 min at 1420g and a 250-µl aliquot of the aqueous methanol layer was added to Sephadex LH-20 minicolumns containing 750 µl of 1.0 N HCl. Columns were swirled and drained and 3 ml of 0.1 N HCl was added, which was eluted to waste (iodide fraction). TH glucuronide fraction was then eluted with 8 ml of H2O and counted in a well gamma detector (Finnson and Eales, 1996). TH glucuronide formation was determined by subtracting cpm of control tubes (⫺UDPGA) from cpm of experimental tubes (⫹UDPGA) and GT activity was expressed as pmol TH glucuronidated/mg protein/h (Finnson and Eales, 1997).
Sulfotransferase (ST) Assay Cytosolic fractions were thawed on ice and diluted (⬃1:25) with 0.1 M Tris–HCl buffer (pH 7.0). A volume of 500 µl of diluted cytosolic fraction was added to siliconized test tubes containing 20 µl of the cofactor 38-phosphoadenosine 58-phosphosulfate (PAPS, Sigma) previously dissolved in buffer (nominal PAPS conc. ⫽ 20 µM). Control tubes received 20 µl of buffer without PAPS. Incubation and extraction procedures were identical to those for the GT assay and the resulting aqueous methanol layer was added to Sephadex LH-20 minicolumns containing 750 µl of 1.0 N HCl. Columns were swirled and drained and 3 ml of 0.1 N HCl was added, which was eluted to waste (iodide). The TH sulfate fraction was then eluted with 8 ml of water and counted in a well gamma detector (Finnson and Eales, 1996). TH sulfate formation was determined by subtracting cpm from control tubes (⫺PAPS) from cpm of experimental tubes (⫹PAPS) and ST activity was expressed as pmol TH sulfated/mg protein/h (Finnson and Eales, 1998).
Deiodination Assay This was based on the method of Shields and Eales (1986). Microsomal fractions were thawed on ice and diluted (⬃1:25) with 0.1 M Tris–HCl buffer (pH ⫽ 7.2) containing 10 mM of DTT and 1 mM of EDTA to achieve a final protein concentration of 0.2–0.8 mg/ml. A volume of 500 µl of diluted microsomal fraction was added to siliconized test tubes and equilibrated in darkness in a water bath (12°C; 140 rpm). The reaction was started by adding 100,000 cpm of either 125Ilabeled T4 or T3 (final conc. ⫽ 1 nM) or rT3 (final conc. ⫽ 1 µM) previously dissolved in 0.1 N NaOH. The reaction was stopped after 60 min by adding 1 ml of chloroform/methanol (2:1 v/v). This mixture was vortexed and centrifuged for 5–10 min at 1420g and a 200-µl aliquot of the aqueous methanol layer was used for HPLC analysis. Deiodination (ORD or IRD) activity was expressed as pmol TH (T4, T3, or rT3 ) deiodinated/mg protein/h.
HPLC Analysis HPLC analyses of deiodination products were conducted with a Gilson IBM binary gradient system using two solvent systems: (A) acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA)/H2O containing 0.1% TFA, ACN gradient increasing linearly from 42 to 54% (10–20 min); and (B) ACN/0.02 M ammonium acetate (pH 4.0), ACN increasing linearly from 47 to 60% (0–25 min) (Sweeting and Eales, 1992a).
Statistics Statistical analyses were performed using either Student’s t test or one-way analysis of variance (ANOVA) followed by Student–Neuman–Kuels test. Levine test was used to confirm homogeneity of variance. Treatments were considered different if P ⬍ 0.05.
RESULTS T3 Treatment T3 treatment decreased HSI (Table 1) and elevated plasma T3 levels but did not alter plasma T4 levels (Fig.
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TABLE 1 Effect of T3 Treatment (Fed a 1% Ration Containing 12 ppm T3 ) and Food Ration (0, 0.5, and 2.0% BW) on Mean (⫾SEM) Body Weight (BW) and Hepatosomatic Index (HSI) of Rainbow Trout Study
Treatment
Weight (g)
HSI (%)
1
Control T3 (12 ppm) Ration 0% Ration 0.5% Ration 2.0%
294 ⫾ 18.7 279 ⫾ 14.3 170 ⫾ 22.6 194 ⫾ 22.9 256 ⫾ 14.7 b
1.4 ⫾ 0.1 1.2 ⫾ 0.1 a 0.7 ⫾ 0.1 0.9 ⫾ 0.1 1.4 ⫾ 0.1 b
2
a HSI value in T -treated trout was significantly lower than in 3 control trout (P ⬍ 0.05). b Body weight and HSI were significantly higher in trout fed a 2.0% BW ration than in trout fed lower (0 and 0.5% BW) rations (P ⬍ 0.05).
1). T3 treatment also decreased hepatic T4ORD activity, increased T4IRD and T3IRD activities but did not change rT3ORD activity (Fig. 2). T3ORD and rT3IRD activities were negligible (data not shown). T3 treatment had no effect on glucuronidation (Fig. 3) or sulfation (Fig. 4) of T4, T3, or rT3.
FIG. 2. Mean (⫾SEM) hepatic T4ORD, T4IRD, T3IRD, and rT3ORD activities of control and T3-treated trout. T4 substrate, 1 nM; T3 substrate, 1 µM. aDifferent from control (P ⬍ 0.05). No T3IRD or rT3IRD activities were detected.
Food Ration Both body weight and HSI were higher in trout fed a 2.0% ration than in trout fed lower (0 and 0.5%) rations
FIG. 1. Mean (⫾SEM) plasma T4 and T3 concentrations of control and T3-treated trout. aHigher than control (P ⬍ 0.05).
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FIG. 3. Mean (⫾SEM) hepatic glucuronidation of T4, T3, and rT3 of control or T3-treated trout. There were no significant differences between treatments (P ⬍ 0.05).
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FIG. 4. Mean (⫾SEM) hepatic sulfation of T4, T3, and rT3 of control and T3-treated trout. There were no significant differences between treatments (P ⬍ 0.05).
(Table 1). Plasma T4 levels were lower in trout fed a 0.5% ration than in trout fed either a 0 or a 2.0% ration but ration had no effect on plasma T3 concentration (Fig. 5). Fasting decreased T4ORD activity and increased rT3ORD activity relative to 0.5 and 2.0%
FIG. 6. Mean (⫾SEM) hepatic T4ORD and rT3ORD activity of trout fed rations of 0, 0.5, or 2.0% BW for 7 days. T4 substrate, 1 nM; rT3 substrate, 1 µM. aSignificantly different 0.5 and 2% (P ⬍ 0.05). No T4IRD, T3ORD, T3IRD, or rT3IRD activities were detected.
rations (Fig. 6). T4IRD, T3ORD, T3IRD, or rT3IRD activities were negligible in trout fed any ration (data not shown). Glucuronidation of T4, T3, and rT3 was greater in trout fed 0 or 0.5% rations than in trout fed 2.0%, and glucuronidation of T3 was greater in starved trout than in trout fed 0.5% (Fig. 7). Sulfation of both T3 and rT3 was greater in trout fed a 2.0% ration than in trout fed 0 and 0.5% rations, ration did not effect T4 sulfation (Fig. 8).
DISCUSSION
FIG. 5. Mean (⫾SEM) plasma T4 and T3 concentrations of trout fed rations 0, 0.5, or 2.0% BW for 7 days. aSignificantly lower than 0 or 2.0% BW (P ⬍ 0.05).
Both hyperthyroidism (T3 treatment) and food ration alter thyroidal status in trout by acting on deiodination pathways (reviewed by Eales and Brown, 1993). Our present goal was to determine if these conditions also influence hepatic TH conjugation pathways, and at the same time we also studied TH deiodination. This allows a comparison to mammalian deiodination and conjugation pathways which are shown to interact (Visser et al., 1990; Visser, 1994). Effects of T3 treatment and food ration on trout hepatic TH conjugation and deiodination are summarized in Table 2.
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384
FIG. 7. Mean (⫾SEM) hepatic glucuronidation of T4, T3, and rT3 of trout fed rations of 0, 0.5, or 2.0% BW for 7 days. aSignificantly lower for 2% than for lower rations (P ⬍ 0.05). bSignificantly lower than for fasted trout (P ⬍ 0.05).
The present responses of trout to experimental T3 hyperthyroidism confirm previous studies in showing reduced hepatic T4ORD activity (< T3 production) and increased IRD activity for both T4 (> rT3 prodution) and T3 (> T2 production) (Eales and Finnson, 1991; Sweeting and Eales, 1992c; MacLatchy and Eales, 1992). All the above changes in hepatic deiodination would contribute to T3 autoregulation. Our novel findings are the negligible effects of T3 hyperthyroidism on glucuronidation or sulfation of T4 , T3 , or rT3 , which also have the potential to contribute to T3 autoregulation. Furthermore, we found no change in high-Km rT3 deiodination, even though formation of rT3 is induced by T3 treatment. Therefore, at least under our experimental conditions, autoregulation of T3 involved modifying only the hepatic low-Km T4ORD, T4IRD, and T3IRD activities. The responses of the trout thyroid system to changes in food ration or fasting confirm previous studies in showing reduced hepatic T4ORD activity following a 7-day fast (Sweeting and Eales, 1992c). The effects of food ration or fasting on trout hepatic T4IRD and T3IRD activities have not been studied previously. Our data show that these inner-ring deiodination pathways are insensitive to food intake. However, fasting did
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Finnson and Eales
FIG. 8. Mean (⫾SEM) hepatic sulfation of T4, T3, and rT3 of trout fed rations of 0, 0.5, or 2.0% BW for 7 days. aSignificantly higher for 2% than for lower rations (P ⬍ 0.05).
increase the hepatic high-Km rT3ORD activity in trout. This suggests a greater need to degrade rT3 during a fast, despite no induction of hepatic rT3 formation. Regardless of the physiological implications, the fasting response of high-Km rT3ORD in the trout is the opposite to that observed in mammals due to type I deiodination (McNabb, 1992). For all TH tested, the highest food ration (2% BW/d) decreased hepatic TH glucuronidation. Glucuronidation is a major pathway for biliary excretion of TH in
TABLE 2 Summary of the Significant (P ⬍ 0.05) Effects of T3 Treatment and Fasting on Outer- and Inner-Ring Deiodination (ORD and IRD), Glucuronidation (Gluc), and Sulfation (Sulf) of TH in Liver of Rainbow Trout Treatment
Pathway
T4
T3
T3-treated
ORD IRD Gluc Sulf ORD IRD Gluc Sulf
⫺ ⫹
⫹
Fasted
⫺ ⫹
rT3
⫹ ⫹ ⫺
⫹ ⫺
Note. Positive (⫹) sign indicates stimulation and negative (⫺) sign indicates inhibition.
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trout (Finnson and Eales, 1996) and so a high food intake would likely decrease biliary TH loss. Reduced biliary loss of T4 and T3 should increase availability of T3 and its T4 precursor. This, coupled with increased hepatic T4ORD activity, should increase T3 availability. Therefore, both depressed TH glucuronidation and increased T4ORD may be complementary in enhancing T3 availability and thyroidal status when food intake is high. This is consistent with the general effect of food intake to elevate thyroidal status in fish. The highest food ration (2% BW/d) increased hepatic sulfation of T3 and rT3 in trout. This is the response opposite to that described above for glucuronide conjugates. Since THS occur in trout bile (Finnson and Eales, 1996), this suggests that the high food ration favors excretion of TH and particularly rT3 as sulfate conjugates. However, the biological significance of sulfation may vary with the type of TH. In contrast to T4S and rT3S, T3S can be desulfated more readily in trout by hepatic mitochondrial/lysosomal and microsomal cell fractions and by isolated hepatocytes to regenerate T3 (Finnson and Eales, 1999). Thus, as suggested for mammals, T3S may act as a temporary inactive lipid-insoluble storage form of T3 that can be reactivated by desulfation (Kung et al., 1988; Santini et al., 1992). Therefore, the enhanced T3 sulfation accompanying the high ration could complement changes in deiodination and glucuronidation to enhance thyroidal status by favoring sequestration of T3 in liver cells. Although both T3 treatment and food intake influence thyroid function, their effects on hepatic TH deiodination and conjugation pathways differ. Therefore the thyroid response to food involves adjustments that do not depend solely on changes in T3 production. There are few studies in other vertebrates on the effects of thyroid status, nutritional status, or other physiological states on TH conjugation. However, in general agreement with our study, Kaptein et al. (1997) showed that rat hepatic sulfation of TH was decreased by fasting but was unaffected by experimental hyperthyroidism (T4 treatment). Gong et al. (1992) showed that rat hepatic sulfation of T3 was regulated by the pattern of pituitary growth hormone secretion. It has also been shown that formation and metabolism of TH conjugation may depend on the stage of development. TH sulfates occur in plasma of fetal lambs (Visser, 1994); Wu et al. (1998) found sulfate conjugates of rT3 and
3,38-T2 in plasma of premetamorphic tadpoles. Further studies on sulfation of TH in different species are needed to assess the role of sulfation in regulating thyroidal status. Under our in vitro assay conditions, both glucuronidation and sulfation showed a substrate preference of rT3 ⬎ T4 ⫽ T3 (Figs. 3 and 4). This is consistent with previous studies on trout (Finnson and Eales, 1997, 1998). Under normal conditions hepatic rT3 production is negligible in trout (Sweeting and Eales, 1992b; Johnston et al., 1996) and rT3 conjugation may therefore be of little physiological consequence. However, under conditions of TH challenge, T4 and T3IRD pathways are induced (Fig. 2) and preferential conjugation and removal of rT3 may then be important. Trout hepatic high-Km rT3ORD activity was unaffected by a T3 challenge but increased during fasting. In both regards the trout high-Km rT3ORD activity differs from rat type I deiodination activity, which increases with hyperthyroidism and decreases during fasting (McNabb, 1992; Kohrle, 1996). Clearly there are major functional differences between trout high-Km rT3ORD and rat type I deiodination. Other differences between these two enzyme systems are summarized by Finnson et al. (1999). We conclude that hepatic TH conjugation pathways are insensitive to experimental T3 hyperthyroidism but do respond to food ration. Both glucuronide and sulfate conjugation may in different ways complement hepatic deiodination in regulating T3 availability and TH homeostasis of trout in different nutritional states. Finally, the high-Km rT3ORD differs from rat type I deiodination in its response to both T3 hyperthyroidism and nutritional state.
ACKNOWLEDGMENTS This study was supported by Natural Sciences and Research Council of Canada Research Grant A1965 to J. G. E.
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