J. Biochem. 112, 112-116 (1992)
Hepatic Triiodothyronine Sulfation and Its Regulation by Growth Hormone and Triiodothyronine in Rats1 Da-Wei Gong, Norie Murayama, Yasushi Yamazoe,2 and Ryuichi Kato Department of Pharmacology, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160 Received for publication, February 10, 1992
The regulatory mechanism of cytosolic sulfation of T3 has been studied in rat liver. Sulfation of T3 is sexually differentiated in adult rats of Sprague-Dawley (SD), Fisher 344, and ACI strains. In SD strain, the male animals showed 4 timeis higher sulfating activity than did the females. The specific activity was decreased by hypophysectomy of male adult rats, but was not affected in the females. Thus, the sex-difference was abolished in the hypophysectomized condition. Supplement of human GH intermittently twice daily for 7 days, to mimic the male secretory pattern, increased T, sulfating activity in both sexes of hypophysectomized rats, whereas continuous infusion to mimic a female secretory pattern had no appreciable effect. Cytosolic sulfation of T3 was decreased by 25 to 30% by thyroidectomy or propylthiouracil treatment of male adult rats, and was restored by the supplementation of T3 (50 /ig/kg daily for 7 days) to thyroidectomized rats. Administration of T3 in hypophysectomized rats almost completely restored the sulfating activity in the males and increased the activity in the females. Cytosolic T3 sulfation was inhibited by the addition of known inhibitors of phenol sulfotransferase, pentachlorophenol or 2,6dichloro-4-nitrophenol. These results indicate a role of pituitary GH in hepatic sulfation of thyroid hormones in rats. The data obtained also raise the possibility that GH may modify the effect of thyroid hormones on the pituitary by a feed-back mechanism through changing the level of a sex-dominant phenol sulfotransferase(s) in rat livers. T3 was also sulfated in hepatic cytosols of mouse, hamster, rabbit, dog, monkey, and human. A sex-related difference was also observed in mice, with the females showing the higher activity.
Sulfate conjugation, occurring mainly in liver, is known to increase the water solubility of chemicals, and thus to facilitate the excretion of lipophilic compounds in urine and feces (1, 2). Main metabolic pathways of iodothyronines are deiodination and conjugation of the phenol group with glucuronide and sulfate (3-5). A series of studies also showed that iodothyronine sulfates are preferred substrates for liver deiodinase: The sulfation precedes and facilitates the microsomal deiodination (6-8). Therefore, sulfation of thyroid hormones has been viewed as an essential step in their metabolism (9). Cytosolic sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to an acceptor (10, 11). In rats, rates of cytosolic sulfation often vary with sex and age: hepatic sulfation of hydroxysteroids, aliphatic amines, and hydroxymethylarenes is female-dominant (12-14), whereas iV-hydroxyarylamine sulfation is male-dominant (15, 16). Previous studies from our laboratory provided evidence that hepatic iV-hydroxy-
arylamine and cortisol sulfations are regulated in rats by pituitary GH (17, 18), whose secretion is pulsatile in males and rather constant in females (29). Thus, sex-specific expression of both sulfations is governed by a sexually differentiated secretory pattern of GH in adult rats. Although sulfation of thyroid hormones has been shown to be catalyzed by a phenol sulfotransferase in rat liver (6, 20), little information is available concerning the hormonal influence on the expression of hepatic sulfotransferase. Thus, we have studied the role of pituitary GH and thyroid hormone on hepatic T3 sulfation of T s in rats. The results obtained indicate that the T3 sulfation is male-dominant, and regulated by the secretory pattern of pituitary GH and T, itself in the rat. MATERIALS AND METHODS Chemicals—3,5-Diiodothyronine (3,5-T2), T,, 3,3',5-triiodo-D-thyronine (D-Ts), 3,3',5'-triiodothyronine (rT 3 ), 3,3',5-triiodothyroacetic acid (TA3), thyroxine (T4), 6-propyl-2-thiouracil (PTU), 3-iodotyrosine, and PAPS were purchased from Sigma Chemical (St. Louis, MO, U.S.A.). ["S]PAPS (91.02 GBq/mmol) was obtained from New England Nuclear (Boston, MA, U.S.A.). 2,6-Dichloro-4nitrophenol (DCNP) and pentachlorophenol (PCP) were obtained from Wako Pure Chemical (Tokyo). Human GH (hGH) was a generous gift from Sumitomo Pharmaceutical (Osaka). Animals and Treatment—Sprague-Dawley (SD), Fisher
1 This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. 2 To whom correspondence should be addressed. Abbreviations:DCNP, 2,6-dichloro-4-nitrophenol; D-T,, 3,3',5-triiodoD-thyronine; GH, growth hormone; HAST, hydroxylamine sulfotransferase; IU, international unit; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; PCP, pentachlorophenol; PTU, 6-propyl-2-thiouracil; rTi, 3,3',5'-triiodothyronine; Tj, 3,3',5-triiodo-L-thyronine; T4, L-thyroxine; TA,, 3,3',5-triiodothyroacetic acid; 3,5-T,, 3,6-diiodothyronine.
112
J. Biochem.
GH and T, Influence of Hepatic T3 Sulfation
113
344 (9 weeks old), and ACI (9 weeks old) rats were obtained from Clea Japan (Tokyo). Liver samples of 3-month-old dwarf rats (21) were kindly supplied by Prof. Ishikawa (Department of Anatomy, School of Medicine, Jikei University, Tokyo). Some rats of SD strain were hypophysectomized or thyroidectomized at 7 weeks of age, and were supplemented with GH or thyroid hormone after 1 week of recovery. hGH was administered by twice-daily injection at 0.2 IU/100 g or by continuous infusion at 0.01 IU/h (22, 23). T, was given by daily subcutaneous injection (5 or 50 n g/kg) for 7 days. PTU was given in drinking water (0.1 mg/ml) for 14 days (24, 25). Cytosols were prepared as described previously (18). Adult BALB/CAnN X DBA/ 2NF1 (CDF1) mice, Hartley guinea pig, and New Zealand white rabbits were obtained from Clea Japan, Tokyo; Syrian golden hamsters were from Tokyo Laboratory Animal Sciences. The source of human livers was the same as described previously (26). Sulfation Assay—Sulfation of T, and its analogs was determined by the method of Sekura et aL (20) with minor modifications: Briefly, the incubation mixture consisted of 250 /zM Tris-HCl (pH 7.9), 5 mM mercaptoethanol, 50 /*M substrate, 200//M "S-PAPS, and 0.5^g/ml cytosolic protein in a total volume of 50//I. The reaction was performed for 20 min at 37'C. The sulfates produced were chromatographed on Kodak cellulose sheet (No. 13225) at 4"C, and quantitated by scintillation counting. Protein was determined by the method of Lowry et ai. (27), and statistics were compiled using Student's ttest. RESULTS Sulfation of T, and Its Regulation by GH and T,— Expression of several enzymes and proteins, including cytochrome P-450 (22, 23, 28-30) and a,u-globulin (32, 32), is now known to be sex-differentiated in rat liver. Rates of cytosolic iV-hydroxyarylamine and hydroxysteroid sulfations are also differ between the sexes (15, 16, 33). Recent studies from our and other laboratories have shown the role of pituitary GH in the sex-dependent expression of 600
Female
H
H
H
GH(8) GH
Hepatic Triiodothyronine Sulfation and Its Regulation by Growth Hormone and Triiodothyronine in Rats1 Da-Wei Gong, Norie Murayama, Yasushi Yamazoe,2 and Ryuichi Kato Department of Pharmacology, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160 Received for publication, February 10, 1992
The regulatory mechanism of cytosolic sulfation of T3 has been studied in rat liver. Sulfation of T3 is sexually differentiated in adult rats of Sprague-Dawley (SD), Fisher 344, and ACI strains. In SD strain, the male animals showed 4 timeis higher sulfating activity than did the females. The specific activity was decreased by hypophysectomy of male adult rats, but was not affected in the females. Thus, the sex-difference was abolished in the hypophysectomized condition. Supplement of human GH intermittently twice daily for 7 days, to mimic the male secretory pattern, increased T, sulfating activity in both sexes of hypophysectomized rats, whereas continuous infusion to mimic a female secretory pattern had no appreciable effect. Cytosolic sulfation of T3 was decreased by 25 to 30% by thyroidectomy or propylthiouracil treatment of male adult rats, and was restored by the supplementation of T3 (50 /ig/kg daily for 7 days) to thyroidectomized rats. Administration of T3 in hypophysectomized rats almost completely restored the sulfating activity in the males and increased the activity in the females. Cytosolic T3 sulfation was inhibited by the addition of known inhibitors of phenol sulfotransferase, pentachlorophenol or 2,6dichloro-4-nitrophenol. These results indicate a role of pituitary GH in hepatic sulfation of thyroid hormones in rats. The data obtained also raise the possibility that GH may modify the effect of thyroid hormones on the pituitary by a feed-back mechanism through changing the level of a sex-dominant phenol sulfotransferase(s) in rat livers. T3 was also sulfated in hepatic cytosols of mouse, hamster, rabbit, dog, monkey, and human. A sex-related difference was also observed in mice, with the females showing the higher activity.
Sulfate conjugation, occurring mainly in liver, is known to increase the water solubility of chemicals, and thus to facilitate the excretion of lipophilic compounds in urine and feces (1, 2). Main metabolic pathways of iodothyronines are deiodination and conjugation of the phenol group with glucuronide and sulfate (3-5). A series of studies also showed that iodothyronine sulfates are preferred substrates for liver deiodinase: The sulfation precedes and facilitates the microsomal deiodination (6-8). Therefore, sulfation of thyroid hormones has been viewed as an essential step in their metabolism (9). Cytosolic sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to an acceptor (10, 11). In rats, rates of cytosolic sulfation often vary with sex and age: hepatic sulfation of hydroxysteroids, aliphatic amines, and hydroxymethylarenes is female-dominant (12-14), whereas iV-hydroxyarylamine sulfation is male-dominant (15, 16). Previous studies from our laboratory provided evidence that hepatic iV-hydroxy-
arylamine and cortisol sulfations are regulated in rats by pituitary GH (17, 18), whose secretion is pulsatile in males and rather constant in females (29). Thus, sex-specific expression of both sulfations is governed by a sexually differentiated secretory pattern of GH in adult rats. Although sulfation of thyroid hormones has been shown to be catalyzed by a phenol sulfotransferase in rat liver (6, 20), little information is available concerning the hormonal influence on the expression of hepatic sulfotransferase. Thus, we have studied the role of pituitary GH and thyroid hormone on hepatic T3 sulfation of T s in rats. The results obtained indicate that the T3 sulfation is male-dominant, and regulated by the secretory pattern of pituitary GH and T, itself in the rat. MATERIALS AND METHODS Chemicals—3,5-Diiodothyronine (3,5-T2), T,, 3,3',5-triiodo-D-thyronine (D-Ts), 3,3',5'-triiodothyronine (rT 3 ), 3,3',5-triiodothyroacetic acid (TA3), thyroxine (T4), 6-propyl-2-thiouracil (PTU), 3-iodotyrosine, and PAPS were purchased from Sigma Chemical (St. Louis, MO, U.S.A.). ["S]PAPS (91.02 GBq/mmol) was obtained from New England Nuclear (Boston, MA, U.S.A.). 2,6-Dichloro-4nitrophenol (DCNP) and pentachlorophenol (PCP) were obtained from Wako Pure Chemical (Tokyo). Human GH (hGH) was a generous gift from Sumitomo Pharmaceutical (Osaka). Animals and Treatment—Sprague-Dawley (SD), Fisher
1 This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. 2 To whom correspondence should be addressed. Abbreviations:DCNP, 2,6-dichloro-4-nitrophenol; D-T,, 3,3',5-triiodoD-thyronine; GH, growth hormone; HAST, hydroxylamine sulfotransferase; IU, international unit; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; PCP, pentachlorophenol; PTU, 6-propyl-2-thiouracil; rTi, 3,3',5'-triiodothyronine; Tj, 3,3',5-triiodo-L-thyronine; T4, L-thyroxine; TA,, 3,3',5-triiodothyroacetic acid; 3,5-T,, 3,6-diiodothyronine.
112
J. Biochem.
GH and T, Influence of Hepatic T3 Sulfation
113
344 (9 weeks old), and ACI (9 weeks old) rats were obtained from Clea Japan (Tokyo). Liver samples of 3-month-old dwarf rats (21) were kindly supplied by Prof. Ishikawa (Department of Anatomy, School of Medicine, Jikei University, Tokyo). Some rats of SD strain were hypophysectomized or thyroidectomized at 7 weeks of age, and were supplemented with GH or thyroid hormone after 1 week of recovery. hGH was administered by twice-daily injection at 0.2 IU/100 g or by continuous infusion at 0.01 IU/h (22, 23). T, was given by daily subcutaneous injection (5 or 50 n g/kg) for 7 days. PTU was given in drinking water (0.1 mg/ml) for 14 days (24, 25). Cytosols were prepared as described previously (18). Adult BALB/CAnN X DBA/ 2NF1 (CDF1) mice, Hartley guinea pig, and New Zealand white rabbits were obtained from Clea Japan, Tokyo; Syrian golden hamsters were from Tokyo Laboratory Animal Sciences. The source of human livers was the same as described previously (26). Sulfation Assay—Sulfation of T, and its analogs was determined by the method of Sekura et aL (20) with minor modifications: Briefly, the incubation mixture consisted of 250 /zM Tris-HCl (pH 7.9), 5 mM mercaptoethanol, 50 /*M substrate, 200//M "S-PAPS, and 0.5^g/ml cytosolic protein in a total volume of 50//I. The reaction was performed for 20 min at 37'C. The sulfates produced were chromatographed on Kodak cellulose sheet (No. 13225) at 4"C, and quantitated by scintillation counting. Protein was determined by the method of Lowry et ai. (27), and statistics were compiled using Student's ttest. RESULTS Sulfation of T, and Its Regulation by GH and T,— Expression of several enzymes and proteins, including cytochrome P-450 (22, 23, 28-30) and a,u-globulin (32, 32), is now known to be sex-differentiated in rat liver. Rates of cytosolic iV-hydroxyarylamine and hydroxysteroid sulfations are also differ between the sexes (15, 16, 33). Recent studies from our and other laboratories have shown the role of pituitary GH in the sex-dependent expression of 600
Female
H
H
H
GH(8) GH
