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Department of Internal Medicine III (E.K., T.J.V.) and Department of Endocrinology and Reproduction (G.A.C.v.H., E.L., W.J.d.G.), Erasmus University Medical School, 3000 DR Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Theo J. Visser, Ph.D., Department of Internal Medicine III, Room Bd 234, Erasmus University Medical School, PO Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: visser{at}inw3.azr.nl
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although sulfation also increases the water solubility of thyroid
hormone, it does not merely serve to facilitate the biliary and/or
urinary excretion of the hormone. Instead, we have shown that sulfation
accelerates the degradation of different iodothyronines by the type I
iodothyronine deiodinase (D1) (4, 5). This enzyme is important for the
peripheral conversion of the prohormone T4 by outer ring deiodination
(ORD) to the active hormone T3 but is also capable of
catalyzing the inner ring deiodination (IRD) of T4 and
T3 to the inactive metabolites rT3 and
3,3'-diiodothyronine (3,3'-T2), respectively (6, 7). The
preferred substrate for this enzyme is rT3, which is very
rapidly converted by ORD to 3,3'-T2. Although sulfation
does not affect the deiodination of rT3, it has dramatic
effects on the deiodination of other iodothyronines (4, 5). The IRD of
T4 by rat D1 is augmented 
200-fold after sulfation of
this substrate, whereas the ORD of T4 sulfate
(T4S) is completely blocked (4, 5). Also the IRD of
T3 sulfate (T3S) is much faster than that of
nonsulfated T3, and this has been observed with human, rat,
and dog D1 (4, 5, 8). In contrast to the inhibited ORD of
T4S, ORD of 3,3'-T2 sulfate
(3,3'-T2S) by rat, dog, and human D1 is extremely fast
(4, 5, 8).
Iodothyronine sulfates are neither deiodinated by the type II deiodinase (D2), which catalyzes the ORD of T4 and rT3, nor by the type III deiodinase (D3), which catalyzes the IRD of T3 and T4 (Refs. 6, 7, and 9 and T. J. Visser and E. Kaptein, unpublished observations). The purpose of the facilitated degradation of T4S and T3S by D1 remains an enigma. It has been speculated that the role of sulfation is especially important when D1 activity is low, i.e. during fetal development and nonthyroidal illness (10). These conditions are associated with dramatic increases in the plasma concentration of the different iodothyronine sulfates (11, 12, 13, 14, 15, 16, 17, 18, 19). In these situations, sulfation is a reversible pathway of thyroid hormone inactivation, since free iodothyronines may be liberated from the conjugates by action of sulfatases expressed in different tissues or by intestinal bacteria (10, 20, 21, 22). Since T3S does not bind to the nuclear T3 receptor, the conjugate is devoid of thyromimetic activity unless it is desulfated (23).
Little is known about the properties of the sulfotransferase isoenzymes catalyzing the sulfation of iodothyronines, let alone about the regulation of their expression. Sekura et al. (24) have studied the sulfation of different iodothyronines by partially purified rat hepatic arylsulfotransferase (AST) I and IV, showing preference for 3,3'-T2 as the substrate. More recent studies of Gong et al. (25), Hurd et al. (26), and Santini et al. (27) have focused on the sulfation of T3 in rat liver cytosol, showing markedly higher hepatic T3 sulfotransferase activities in male than in female animals. This was correlated with the sex-dependent pattern of GH secretion in rats, i.e. pulsatile in males and more constant in females (25). T3 sulfation has also been demonstrated in rat brain and kidney (26) as well as in human liver and intestine (28, 29). In the present study, hepatic iodothyronine sulfotransferase activity was characterized in male and female rats using 3,3'-T2 as the preferred substrate, and occasionally with T3 as the substrate. In addition, the possible effects of hypothyroidism, hyperthyroidism, short-term fasting, and long-term food restriction on hepatic iodothyronine sulfotransferase activities in both sexes were determined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Male and female Wistar rats were obtained from Harlan
Sprague-Dawley (Zeist, The Netherlands) or bred locally. They were
housed in a controlled animal room with a 14-h light, 10-h dark
photocycle and were provided ad libitum with food and
drinking water. All experiments, which have also been described
previously (30, 31), were approved by the Animal Welfare Committee
(DEC) of Erasmus University.
Thyroid state. Rats were made hypothyroid by treatment for 2 weeks with drinking water containing 0.1% (wt/vol) methimazole. Hyperthroidism was induced by treating rats for 7 days with daily ip injections of 10 µg T4 per 100 g body wt, while control rats received injections with vehicle (30).
Nutrition state. At the start of the experiments, rats were 10 weeks old (mean body wt: male rats, 216 g; female rats, 163 g). Daily food intake of control rats was 24 g in males and 15 g in females. Acute effects of starvation were studied in rats completely deprived of food for 3 days. The long-term effects of food restriction were studied in rats that were provided with only one-third of normal food intake during 3 weeks (FR33), i.e. 8 g for males and 5 g for females. Control animals continued to have free access to food, and all animals were supplied with drinking water ad libitum (31).
At the end of the treatments (24 h after the last T4 dose), rats were anesthetized with ether and decapitated. Livers were isolated, immediately frozen in liquid nitrogen, and stored at -80 C until further processing. Liver tissue was homogenized in 0.25 M sucrose, 10 mM HEPES, and 1 mM dithiothreitol, and cytosol was prepared and stored in aliquots at -80 C as previously described (30, 31). Protein was measured with the Bio-Rad protein assay (Bio-Rad, Veenendaal, The Netherlands) using BSA as the standard.
Sulfotransferase assay
Iodothyronine sulfotransferase activities were usually assayed
by incubation of 1 µM 3,3'-T2 or
T3 and 100,000 cpm of the 125I-labeled compound
for 30 min at 37 C with the indicated amounts of liver cytosol in the
presence or absence (blank) of 50 µM PAPS in 0.2 ml 0.1
M phosphate (pH 7.2) and 2 mM EDTA. Identical
results were obtained using phosphate buffer without EDTA or buffer
containing 2 mM Mg2+ or Ca2+. The
reactions were started by addition of cytosol diluted in ice-cold
buffer and stopped by addition of 0.8 ml 0.1 M HCl. The
mixtures were applied to Sephadex LH-20 minicolumns (bed volume, 1 ml),
equilibrated in 0.1 M HCl. Iodide, sulfated iodothyronines,
and nonsulfated iodothyronines were successively eluted with 2x 1 ml
0.1 M HCl, 6x 1 ml ethanol/water (20/80, vol/vol), and 3x
1 ml ethanol/0.1 M NaOH (50:50, vol/vol), respectively.
Fractions were collected and counted for radioactivity. Sulfation in
complete reaction mixtures was corrected for minor radioactivity
detected in the corresponding fractions of the blanks. The use of
special racks for parallel collection of fractions from 16 columns and
a 16-channel 
-counter, with processing of the data by computer,
allowed the analysis of a large number of samples in a single
experiment.
Statistical analysis
Results are presented as means ±SD or as means of
triplicate determinations in a representative experiment. Where
appropriate, differences between groups were evaluated statistically by
unpaired or paired Student’s t test or by ANOVA followed by
Duncan’s multiple range test.
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Results |
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shows the chromatography of
acidified reaction mixtures after incubation of radioactive
3,3'-T2 with male rat liver cytosol in the absence or
presence of PAPS. After incubation without PAPS, no radioactivity was
eluted from the Sephadex minicolumns with acidic (0.1 M
HCl) or neutral (20% ethanol in water) solvent but only with alkaline
solvent (50% ethanol in 0.1 M NaOH), typical for the
chromatography of nonsulfated 3,3'-T2 (32). After
incubation in the presence of PAPS, substantial radioactivity was
eluted with the neutral solvent, peaking in the same fractions as
synthetic 3,3'-T2S (32). More than 95% of applied
3,3'-T2S was recovered in the neutral fractions. Similar
separation was obtained between T3 and T3S (not
shown). This method is generally applicable for separation of free and
conjugated iodothyronines and is also used for fractionation of
iodothyronine glucuronyltransferase assay mixtures (33). In the absence
of added PAPS, no glucuronidation of 3,3'-T2 was observed,
which is not surprising since the uridine
diphosphate-glucuronyltransferases are located in the microsomes, and
no cofactor (uridine diphosphate-glucuronic acid) was added (33).
Neither in the absence nor in the presence of PAPS was any radioiodide
formation observed even though 3,3'-T2S is a good substrate
for D1, indicating that D1 activity is not present in rat liver cytosol
but only in the microsomal fraction (6, 7).
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compares the effects of
increasing concentrations of unlabeled 3,3'-T2 and
T3 on the sulfation of radioactive 3,3'-T2 and
T3 by male rat liver cytosol in the presence of PAPS.
Although the rate of T3 sulfation was much lower than that
of 3,3'-T2, the dose-inhibition curves for unlabeled
3,3'-T2 and T3 were very similar if their
effects on the sulfation of radioactive 3,3'-T2 or
T3 were compared. In both cases, IC50 values
were at least 10-fold lower for 3,3'-T2 than for
T3. These results suggest that 3,3'-T2 and
T3 are substrates for the same sulfotransferase
isoenzyme(s), which is (are) more readily saturated by
3,3'-T2 than by T3.
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shows the sulfation of
3,3'-T2 by male and female rat liver cytosol in the
presence of PAPS as a function of incubation time and cytosolic protein
concentration. Under the same conditions, 3,3'-T2 was
sulfated more rapidly in male than in female rat liver. Regardless of
gender, 3,3'-T2 sulfation was linear with incubation time
until 30% of the substrate was converted (Fig. 3A). With longer
incubation times, sulfation rates leveled off probably due to substrate
depletion. Since PAPS was added in large excess, depletion of the
cofactor is unlikely. In both male and female rat liver,
3,3'-T2 sulfation initially showed a more than proportional
increase with the cytosolic protein concentration (Fig. 3B). For
instance, an increase in the cytosolic protein concentration from 10 to
25 µg/ml resulted in a 4-fold increase in 3,3'-T2S
formation in both males and females. At higher protein concentrations,
3,3'-T2 sulfation appeared to increase linearly with the
protein concentration until significant substrate depletion
occurred.
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presents the effects of pH on
the sulfation of 3,3'-T2 by male and female rat liver
cytosol in the presence of PAPS. At all pH values, the rate of
3,3'-T2 sulfation was markedly higher in male than in
female rat liver. In both sexes, highest 3,3'-T2 sulfation
rates were observed at pH 8. However, all subsequent experiments were
carried out at the more physiological pH value of 7.2, providing
sulfation rates that were 70% of those at the optimal pH.
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shows the sulfation of
3,3'-T2 by male and female rat liver cytosol at varying
3,3'-T2 concentrations (0.5–10 µM) and a
fixed PAPS concentration (50 µM). At all
3,3'-T2 concentrations, sulfation rates were greater in
male than in female rat liver. In both sexes, sulfation demonstrated
saturation kinetics in the range of the 3,3'-T2
concentrations tested. The double-reciprocal plots of sulfation rates
vs. 3,3'-T2 concentration were linear, allowing
the calculation of apparent Km values for
3,3'-T2 and Vmax values (at 50 µM
PAPS). Table 1 presents the kinetic
parameters derived from four experiments, showing that apparent
Km values for 3,3'-T2 were significantly lower
and Vmax values somewhat higher in male than in female rat
liver. From the data shown in Fig. 2
, an apparent Km value
of 48 µM and Vmax value of 0.22 nmol/min per
mg protein were calculated for T3 sulfation by male rat
liver cytosol in the presence of 50 µM PAPS. Therefore,
the apparent Km value for T3 is 30-fold
higher and the Vmax value 10-fold lower than the
corresponding values for 3,3'-T2 sulfation.
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shows the effects of increasing
PAPS concentrations (1–25 µM) on the sulfation of 1
µM 3,3'-T2 by male and female rat liver
cytosol. At all PAPS concentrations, 3,3'-T2 sulfation
rates were higher in male than in female rat liver cytosol. In both
sexes, 3,3'-T2 sulfation approached maximum rates at PAPS
concentrations above 10 µM. The Lineweaver-Burk plots
of these data were linear, from which apparent Km values
for PAPS and Vmax values (at 1 µM
3,3'-T2) were calculated. The kinetic parameters from three
such experiments are presented in Table 1, showing that the apparent
Km value for PAPS is slightly higher while the
Vmax value is markedly higher in male than in female rat
liver.
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presents the effects of
thyroid state on hepatic 3,3'-T2 sulfotransferase
activities in male and female rats. The hypothyroid state of the
methimazole-treated rats was demonstrated by marked decreases in serum
T4 and T3 levels, as well as in hepatic D1
activities, and strong increases in serum TSH levels (30). Conversely,
the hyperthyroid animals showed large increases in serum T4
and T3 levels, as well as in hepatic D1 activities, and
marked decreases in serum TSH levels. In euthyroid controls, hepatic
3,3'-T2 sulfotransferase activity was 2.5 times higher
in males than in females. Methimazole-induced hypothyroidism was
associated with a significant, 28% decrease in sulfotransferase
activity in males but had no effect in females. Hyperthyroidism
slightly decreased sulfotransferase activity by 12% in male rats but
had no effect in female rats.
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shows that short-term fasting did not
affect hepatic 3,3'-T2 sulfotransferase activities in
either males or females. A similar lack of effect of short-term fasting
was observed if hepatic iodothyronine sulfotransferase activity was
determined using T3 as the substrate (not shown).
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shows the effects of FR33 on hepatic
iodothyronine sulfotransferase activities determined with
3,3'-T2 and T3 as substrates. Food restriction
resulted in a large, 51% decrease in 3,3'-T2
sulfotransferase activity in male rats but had no effect in female
rats, so that values were no longer different between food-restricted
males and females (Fig. 9A). The sex-dependent difference in hepatic
iodothyronine sulfotransferase activities in fed controls was even
greater with T3 (4.0-fold) than with 3,3'-T2
(2.1-fold) as substrate. Long-term food restiction also resulted in a
marked, 40% reduction in hepatic T3 sulfotransferase
activity in male rats but was without any effect in female rats.
T3 sulfation remained somewhat higher in food-restricted
males than in females (Fig. 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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200 times higher for
3,3'-T2 than for T3. Hurd et al.
(26) reported a somewhat higher Km value (114
µM) for T3 sulfation by male rat liver
cytosol and a much lower Vmax value (0.16 nmol/h per mg
protein). This may be explained, at least in part, by the use of a much
lower PAPS concentration (0.4 µM) compared with our
experiments (50 µM). We found higher hepatic iodothyronine sulfotransferase activities with both 3,3'-T2 and T3 as substrate in male than in female rats. This is in agreement with previous findings reported by others (25, 26, 27). However, Gong et al. (25) showed that the sex dependence of hepatic sulfotransferase activity varies among species. Opposite to the situation in rats, hepatic T3 sulfotransferase activity is higher in female than in male mice, whereas no sex dependence is observed in humans. Gong et al. (25) have demonstrated that the higher T3 sulfotransferase activity in male vs. female rat liver is not directly dependent on sex hormones. Instead, they found that this is determined by the different GH secretion patterns, being pulsatile in male rats and more constant in female rats. The group of Yamazoe (37) also provided evidence that the sex-dependent expression of certain cytochrome P450 isoenzymes in rat liver is also regulated by this difference in GH secretion pattern. The higher hepatic iodothyronine sulfotransferase activity in male vs. female rats is associated with higher serum levels of different iodothyronine sulfates in male than in female rats. This is not only true for the basal serum levels but in particular also for the increased serum levels of these conjugates observed in rats with impaired D1 activity due to selenium deficiency (38).
We found that the higher 3,3'-T2 sulfotransferase activity in male than in female rat liver is associated with a small increase in Vmax value as well as a larger decrease in apparent Km value for 3,3'-T2. These findings probably do not reflect true differences in Km values, e.g. due to enzyme modification, but presumably represent differences in sulfotransferase isoenzyme composition. We and others have demonstrated that rSULT1B1 and rSULT1C1 are important isoenzymes for the sulfation of iodothyronines in rat liver, whereas rSULT1A1 does not catalyze iodothyronine sulfation (34, 35, 36). Expression of rSULT1C1 is much higher in male than in female rat liver, which has also been ascribed to the different GH secretion patterns in male and female rats (39, 40). In contrast, rSULT1B1 expression in rat liver appears to be independent of gender (34, 35). Therefore, sulfation of T3 and 3,3'-T2 in female rat liver probably represents predominantly the activity of rSULT1B1, whereas sulfation of these iodothyronines in male rat liver is catalyzed in addition by rSULT1C1. This suggests that the apparent Km value for 3,3'-T2 sulfation in female rat liver cytosol (4.2 µM) largely reflects the Km value of rSULT1B1, whereas the apparent Km value for 3,3'-T2 in male rat liver cytosol (1.8 µM) represents a composite value, intermediate between the Km values of rSULT1B1 and rSULT1C1. This is in agreement with our finding that the Km value for 3,3'-T2 sulfation by recombinant rSULT1C1 amounts to 0.75 µM (36); the Km value for recombinant rSUL1B1 has not yet been determined. It should be mentioned that sulfotransferases may consist not only of two identical subunits but also of two different subunits (41). Dependence of sulfotransferase activity on homo- or heterodimer formation may explain our finding of a more than linear increase in 3,3'-T2 sulfation rate with the cytosolic protein concentration.
Gong et al. (25) reported an increase in hepatic T3 sulfotransferase activity in hyperthyroid male rats, whereas Hurd et al. (26) found no difference between normal and hyperthyroid animals. We did not observe an increase in hepatic 3,3'-T2 sulfotransferase activity in hyperthyroid rats, although we found that hypothyroidism results in a significant decrease in males but not in females. In contrast to the lack of effect of short-term fasting on hepatic T3 and 3,3'-T2 sulfotransferase activities in both male and female rats, we observed a marked decrease in sulfotransferase activities for both substrates after long-term food restriction in males but not in females. Both hypothyroidism and food deprivation are known to be associated with a decreased GH secretion (42), where the effect of food deprivation may be mediated, at least in part, by the hypothyroid state of the (semi)starved animals. We therefore speculate that the male-specific decrease in hepatic iodothyronine sulfotransferase activity by both hypothyroidism and long-term food restriction is due to diminished expression of rSULT1C1 secondary to impaired GH secretion. Apparently, 3 days of fasting is not sufficient to produce a significant decrease in sulfotransferase expression.
In conclusion, we have developed a convenient method for the analysis of iodothyronine sulfotransferase activity in tissue cytoplasmic fractions. In agreement with previous reports, we found that this activity is higher in male than in female rat liver. We demonstrate that hepatic sulfation of thyroid hormone is not affected by hyperthyroidism and short-term fasting, whereas it is decreased by hypothyroidism and long-term food restriction in male but not in female rats. We speculate that the latter effects are mediated by an impaired GH secretion, resulting in diminished expression of the male-dominant isoenzyme rSULT1C1.
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Acknowledgments |
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Footnotes |
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2 Present address: Fondation pour Récherches Medicales,
Université de Genève, CH-1211 Genève 4,
Switzerland.
Received May 19, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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