Sulfation of Iodothyronines by Recombinant Human Liver Steroid Sulfotransferases

doi:10.1006/bbrc.1999.1419

Biochemical and Biophysical Research Communications

Volume 263, Issue 3, 5 October 1999, Pages 632-639

https://doi.org/10.1006/bbrc.1999.1419 Get rights and content

Abstract

Sulfation is an important pathway in the metabolism of thyroid hormones. Sulfated iodothyronines are elevated in nonthyroidal illnesses and in the normal human fetal circulation. We assayed and characterized COS-1 cell expressed recombinant human liver dehydroepiandrosterone sulfotransferase (DHEA ST or SULT2A1) and estrogen sulfotransferase (EST or SULT1E1) activities for the first time with triiodothyronine (T₃) as the substrate. Several biochemical properties that included apparent K_m values, thermal stabilities, and responses to the inhibitors 2,6-dichloro-4-nitrophenol and NaCl were tested. SULT2A1, a member of the hydroxysteroid sulfotransferase family, used 3,3′-T₂ more readily than T₃ and 3,5-T₂ as substrates, but had the lowest apparent K_m value for T₃ of any reported human SULT. SULT1E1, a member of the phenol sulfotransferase family, used 3,3′-T₂ and rT₃ more readily than T₃, and also displayed the greatest specificity for T₄ among human SULTs. SULT2A1 may contribute more to iodothyronine sulfation than previously suspected. Potential roles of both steroid sulfotransferases in the enhanced sulfation of nonthyroidal illnesses and fetal development invite further investigation.

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Human liver cytosolic sulfotransferase 2A1-dependent dehydroepiandrosterone sulfation assay by ultra-high performance liquid chromatography-tandem mass spectrometry
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Sulfotransferase 2A1 (SULT2A1) is a major catalyst of the sulfation of dehydroepiandrosterone (DHEA) to dehydroepiandrosterone sulfate (DHEA-S) in human liver cytosol. However, there is a lack of a sensitive and fast analytical method for the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. Therefore, we developed and validated an ultra-high performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method to quantify DHEA-S and used it to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. DHEA-S and cortisol (internal standard) eluted at 2.95 and 2.75 min, respectively. Negative multiple reaction monitoring was used to quantify DHEA-S (m/z 367.3 → 97.0) and cortisol (m/z 407.2 → 331.3). No interfering peaks were observed in blank samples. The lower limit of quantification was 0.2 pmol DHEA-S and the calibration curve was linear from 0.2 to 200 pmol. The intra-day and inter-day accuracy and precision was <11.7%. DHEA-S in the quality control samples was stable at room temperature, 4 °C, and −20 °C. The cytosolic matrix (20–100 μg cytosolic protein) did not affect DHEA-S quantification. Our UPLC–MS/MS method was applied to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. The optimal levels of MgCl₂ and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) cofactor were 2.5 mM and 20 μM, respectively. Reducing agents, including 2-mercaptoethanol and DL-dithiothreitol, did not affect the enzyme activity. A linear relationship existed between DHEA sulfation and amount of human liver cytosol (20–200 μg cytosolic protein) or incubation time (5–30 min). This UPLC–MS/MS approach is safer, easier, and faster than existing radiometric-based sulfotransferase enzyme assays, and it is the first UPLC–MS/MS method for determining SULT2A1-dependent DHEA sulfation in human liver cytosol.
NPY and MC4R signaling regulate thyroid hormone levels during fasting through both central and peripheral pathways
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Fasting-induced suppression of the hypothalamic-pituitary-thyroid (HPT) axis is an adaptive response to decrease energy expenditure during food deprivation. Previous studies demonstrate that leptin communicates nutritional status to the HPT axis through thyrotropin-releasing hormone (TRH) in the paraventricular nucleus (PVN) of the hypothalamus. Leptin targets TRH neurons either directly or indirectly via the arcuate nucleus through pro-opiomelanocortin (POMC) and agouti-related peptide/neuropeptide Y (AgRP/NPY) neurons. To evaluate the role of these pathways in vivo, we developed double knockout mice that lack both the melanocortin 4 receptor (MC4R) and NPY. We show that NPY is required for fasting-induced suppression of Trh expression in the PVN. However, both MC4R and NPY are required for activation of hepatic pathways that metabolize T₄ during the fasting response. Thus, these signaling pathways play a key role in the communication of fasting signals to reduce thyroid hormone levels both centrally and through a peripheral hepatic circuit.
Sulfation of tibolone metabolites by human postmenopausal liver and small intestinal sulfotransferases (SULTs)
2006, Steroids
Sulfation is a major pathway in humans for the biotransformation of steroid hormones and structurally related therapeutic agents. Tibolone is a synthetic steroid used for the treatment for climacteric symptoms and postmenopausal osteoporosis. Sulfation inactivates the hydroxylated metabolites, 3α-hydroxytibolone (3α-OH-tibolone) and 3β-hydroxytibolone (3β-OH-tibolone), and contributes to the regulation of tissue responses to tibolone. We detected SULT1A1, SULT1A3, SULT1E1 and SULT2A1 mRNA expression by RT-PCR in postmenopausal liver and small intestine. Liver pool (n = 5) SULT activities measured with tibolone substrates reflected COS-1 expressed SULT2A1 and SULT1E1 activities. Liver SULT2A1 activity (1.8 ± 0.3 units/mg protein, n = 8, mean ± SEM), and activities with 3α-OH-tibolone (0.6 ± 0.1, n = 8) and 3β-OH-tibolone (0.9 ± 0.2, n = 8) were higher than SULT1E1 activities (<0.05, n = 10). SULT1E1 activities were low or not detected in many samples. Mean small intestinal activities were 0.03 ± 0.01 with 3α-OH-tibolone and 0.04 ± 0.01 with 3β-OH-tibolone (n = 3). In conclusion, SULT2A1 is the major endogenous enzyme responsible for sulfation of the tibolone metabolites in human postmenopausal tissues. The results support the occurrence of pre-receptor enzymatic regulation of hydroxytibolone metabolites and prompt further investigation of the tissue-selective regulation of tibolone effects.
The Nuclear Receptor CAR Is a Regulator of Thyroid Hormone Metabolism during Caloric Restriction
2004, Journal of Biological Chemistry
The orphan nuclear receptor CAR (NR1I3) has been characterized as a central component in the coordinate response to xenobiotic and endobiotic stress. In this study, we demonstrate that CAR plays a pivotal function in energy homeostasis and establish an unanticipated metabolic role for this nuclear receptor. Wild-type mice treated with the synthetic CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) exhibited decreased serum concentration of the thyroid hormone (TH) thyroxine (T₄). However, treatment of Car^–/– mice with TCPOBOP failed to elicit these changes. To examine whether CAR played a role in the regulation of TH levels under physiological conditions, wild-type and Car^–/– mice were fasted for 24 h, a process known to alter TH metabolism in mammals. As expected, the serum triiodothyronine and T₄ concentrations decreased in wild-type mice. However, triiodothyronine and T₄ levels in fasted Car^–/– mice remained significantly higher than those in fasted wild-type animals. Concomitant with the changes in serum TH levels, both CAR agonist treatment and fasting induced the expression of CAR target genes (notably, Cyp2b10, Ugt1a1, Sultn, Sult1a1, and Sult2a1) in a receptor-dependent manner. Importantly, the Ugt1a1, Sultn, Sult1a1, and Sult2a1 genes encode enzymes that are capable of metabolizing TH. An attenuated reduction in TH levels during fasting, as observed in Car^–/– mice, would be predicted to increase weight loss during caloric restriction. Indeed, when Car^–/– animals were placed on a 40% caloric restriction diet for 12 weeks, Car^–/– animals lost over twice as much weight as their wild-type littermates. Thus, CAR participates in the molecular mechanisms contributing to homeostatic resistance to weight loss. These data imply that CAR represents a novel therapeutic target to uncouple metabolic rate from food intake and has implications in obesity and its associated disorders.
Sulfation of iodothyronines by human sulfotransferase 1C1 (SULT1C1)
2000, Biochemical Pharmacology
Citation Excerpt :
The 50% inactivation temperature of SULT1C1 was 38.3°. This activity was more thermolabile than SULT1E1 and SULT2A1 activities when assayed with T3[9]. DCNP and NaCl are known inhibitors of SULTs [4, 9, 19].
Sulfation is an important component of human thyroid hormone metabolism. The role of the human sulfotransferase 1C1 (SULT1C1) is not known. Because SULT1C1 is present in the adult thyroid, intra-thyroidal sulfation of thyroid hormones and their metabolites might occur. We tested this hypothesis by determining the ability of recombinant human SULT1C1 to catalyze iodothyronine sulfation. Apparent K_m values for 3,3′,5-triiodothyronine (T₃), 3,3′-diiodothyronine (3,3′-T₂), 3′,5′,3-triiodothyronine (rT₃), and 3,3′,5,5′-tetraiodothyronine (T₄) with SULT1C1 were 28.7, 10.3, 10.2, and 59.3 μM, respectively. Thermal stability and responses to inhibitors also were tested with T₃ as the substrate. Enzyme aliquots were measured simultaneously to determine SULT1C1 substrate preferences at optimal iodothyronine concentrations. SULT1C1 activity obtained with T₃ was used as 100%, and the activities with 3,3′-T₂, rT₃, T₄, and 3,5-diiodothyronine (3,5-T₂) were 614, 314, 25, and 4%, respectively. We report for the first time the characterization of human SULT1C1 with T₃ and the preferences of the enzyme for various iodothyronines. The presence of SULT1C1 in the adult thyroid gland raises the possibilities that the enzyme can contribute to intraglandular thyroid hormone processing and iodide reutilization.
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¹: To whom correspondence and reprint requests should be addressed at Section of Endocrinology, Diabetes and Metabolism, VAMC, Research 151, 4101 Woolworth Avenue, Omaha, NE 68105. Fax: 402-977-5624. E-mail: [email protected].

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Regular ArticleSulfation of Iodothyronines by Recombinant Human Liver Steroid Sulfotransferases

Abstract

Trends Endocrinol. Metab.

J. Steroid Biochem. Mol. Biol.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

Anal. Biochem.

Anal. Biochem.

Biochem. Biophys. Res. Commun.

Chem. Biol. Interact.

Endocrinology

J. Clin. Endocrinol. Metab.

J. Clin. Endocrinol. Metab.

J. Clin. Endocrinol. Metab.

Pediatr. Res.

Thyroid

J. Clin. Endocrinol. Metab.

J. Biochem.

Mol. Pharmacol.

FASEB J.

Regular Article
Sulfation of Iodothyronines by Recombinant Human Liver Steroid Sulfotransferases