32 research outputs found
Differential expression and ciprofibrate induction of hepatic UDP-glucuronyltransferases for thyroxine and triiodothyronine in Fischer rats
Characterization of iodothyronine sulfotransferase activity in rat liver
Sulfation is an important pathway in the metabolism of thyroid hormone
because it strongly facilitates the degradation of the hormone by the type
I iodothyronine deiodinase. However, little is known about the properties
and possible regulation of the sulfotransferase(s) involved in the
sulfation of thyroid hormone. We have developed a convenient method for
the analysis of iodothyronine sulfotransferase activity in tissue
cytosolic fractions, using radioiodinated 3,3'-diiodothyronine (3,3'-T2)
as the preferred substrate, unlabeled
3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfate donor, and
Sephadex LH-20 minicolomns for separation of the products. We found that
iodothyronine sulfotransferase activity in rat liver cytosol is 1) higher
in male than in female rats; 2) optimal at pH 8.0; 3) characterized (at 50
microM PAPS and pH 7.2) by apparent Michaelis-Menton (Km) values for
3,3'-T2 of 1.77 and 4.19 microM, and Vmax values of 1.94 and 1.45 nmol/min
per mg protein in male and female rats, respectively; 4) characterized (at
1 microM 3,3'-T2 and pH 7.2) by apparent Km values for PAPS of 4.92 and
3.80 microM and Vmax values of 0.72 and 0.31 nmol/min per mg protein, in
males and females, respectively; 5) little affected by hyperthyroidism in
both male and female rats, but significantly decreased by hypothyroidism
in males but not in females; and 6) not affected by short-term (3 days)
fasting in both male and female rats, but significantly decreased by
long-term (3 weeks) food restriction to one-third of normal intake in
males but not in females. It is suggested that the higher hepatic
iodothyronine sulfotransferase activity in male vs. female rats, as well
as the decreases induced in males by hypothyroidism and long-term food
restriction, represents differences in the expression of the male-dominant
isoenzyme rSULT1C1
Thyroid function and deiodinase activities in rats with marginal iodine deficiency
The hypothesis tested was whether marginal iodine deficiency for a period of 6 wk affects iodothyronine deiodinase activities in liver and brain of rats. Male rats were fed purified diets either deficient or sufficient in iodine; the diets were fed on a restricted basis (60% of ad libitum intake). Body weight gain of the two groups was comparable. Iodine deficiency was evidenced by increased thyroid weight (26%), reduced urinary iodine excretion (80%), and reduced plasma T4 concentrations (22%). Activities of liver type I and brain type III deiodinase were unchanged, but the activity of type II deiodinase in brain was increased (28%) in the iodine-deficient rats. Food restriction per se significantly lowered T3 (30%) and T4 (22%) concentrations in plasma and decreased type III deiodinase activity in brain (30%). These results indicate that in marginal iodine deficiency the activities of hepatic type I deiodinase and brain type III deiodinase are unchanged, whereas that of brain type II deiodinase is increased
Expression of chicken hepatic type I and type III iodothyronine deiodinases during embryonic development
In embryonic chicken liver (ECL) two types of iodothyronine deiodinases
are expressed: D1 and D3. D1 catalyzes the activation as well as the
inactivation of thyroid hormone by outer and inner ring deiodination,
respectively. D3 only catalyzes inner ring deiodination. D1 and D3 have
been cloned from mammals and amphibians and shown to contain a
selenocysteine (Sec) residue. We characterized chicken D1 and D3
complementary DNAs (cDNAs) and studied the expression of hepatic D1 and D3
messenger RNAs (mRNAs) during embryonic development. Oligonucleotides
based on two amino acid sequences strongly conserved in the different
deiodinases (NFGSCTSecP and YIEEAH) were used for reverse
transcription-PCR of poly(A+) RNA isolated from embryonic day 17 (E17)
chicken liver, resulting in the amplification of two 117-bp DNA fragments.
Screening of an E17 chicken liver cDNA library with these probes led to
the isolation of two cDNA clones, ECL1711 and ECL1715. The ECL1711 clone
was 1360 bp long and lacked a translation start site. Sequence alignment
showed that it shared highest sequence identity with D1s from other
vertebrates and that the coding sequence probably lacked the first five
nucleotides. An ATG start codon was engineered by site-directed
mutagenesis, generating a mutant (ECL1711M) with four additional codons
(coding for MGTR). The open reading frame of ECL1711M coded for a
249-amino acid protein showing 58-62% identity with mammalian D1s. An
in-frame TGA codon was located at position 127, which is translated as Sec
in the presence ofa Sec insertion sequence (SECIS) identified in the
3'-untranslated region. Enzyme activity expressed in COS-1 cells by
transfection with ECL1711M showed the same catalytic, substrate, and
inhibitor specificities as native chicken D1. The ECL1715 clone was 1366
bp long and also lacked a translation start site. Sequence alignment
showed that it was most homologous with D3 from other species and that the
coding sequence lacked approximately the first 46 nucleotides. The deduced
amino acid sequence showed 62-72% identity with the D3 sequences from
other species, including a putative Sec residue at a corresponding
position. The 3'-untranslated region of ECL1715 also contained a SECIS
element. These results indicate that ECL1711 and ECL1715 are
near-full-length cDNA clones for chicken D1 and D3 selenoproteins,
respectively. The ontogeny of D1 and D3 expression in chicken liver was
studied between E14 and 1 day after hatching (C1). D1 activity showed a
gradual increase from E14 until C1, whereas D1 mRNA level remained
relatively constant. D3 activity and mRNA level were highly significantly
correlated, showing an increase from E14 to E17 and a strong decrease
thereafter. These results suggest that the regulation of chicken hepatic
D3 expression during embryonic development occurs predominantly at the
pretranslational level
Characterization of a propylthiouracil-insensitive type I iodothyronine deiodinase
Mammalian type I iodothyronine deiodinase (D1) activates and inactivates
thyroid hormone by outer ring deiodination (ORD) and inner ring
deiodination (IRD), respectively, and is potently inhibited by
propylthiouracil (PTU). Here we describe the cloning and characterization
of a complementary DNA encoding a PTU-insensitive D1 from teleost fish
(Oreochromis niloticus, tilapia). This complementary DNA codes for a
protein of 248 amino acids, including a putative selenocysteine (Sec)
residue, encoded by a TGA triplet, at position 126. The 3' untranslated
region contains two putative Sec insertion sequence (SECIS) elements.
Recombinant enzyme expressed in COS-1 cells catalyzes both ORD of T4 and
rT3 and IRD of T3 and T3 sulfate with the same substrate specificity as
native tilapia D1 (tD1), i.e. rT3 >> T4 > T3 sulfate > T3. Native and
recombinant tD1 show equally low sensitivities to inhibition by PTU,
iodoacetate, and gold thioglucose compared with the potent inhibitions
observed with mammalian D1s. Because the residue 2 positions downstream
from Sec is Pro in tD1 and in all (PTU-insensitive) type II and type III
iodothyronine deiodinases but Ser in all PTU-sensitive D1s, we prepared
the Pro128Ser mutant of tD1. The mutant enzyme showed strongly decreased
ORD and somewhat increased IRD activity, but was still insensitive to PTU.
These results provide new information about the structure-activity
relationship of D1 concerning two characteristic properties, i.e.
catalysis of both ORD and IRD, and inhibition by PTU
Reduced activation and increased inactivation of thyroid hormone in tissues of critically ill patients
Critical illness is often associated with reduced TSH and thyroid hormone
secretion as well as marked changes in peripheral thyroid hormone
metabolism, resulting in low serum T(3) and high rT(3) levels. To study
the mechanism(s) of the latter changes, we determined serum thyroid
hormone levels and the expression of the type 1, 2, and 3 iodothyronine
deiodinases (D1, D2, and D3) in liver and skeletal muscle from deceased
intensive care patients. To study mechanisms underlying these changes, 65
blood samples, 65 liver, and 66 skeletal muscle biopsies were obtained
within minutes after death from 80 intensive care unit patients randomized
for intensive or conventional insulin treatment. Serum thyroid parameters
and the expression of tissue D1-D3 were determined. Serum TSH, T(4), T(3),
and the T(3)/rT(3) ratio were lower, whereas serum rT(3) was higher than
in normal subjects (P < 0.0001). Liver D1 activity was down-regulated and
D3 activity was induced in liver and skeletal muscle. Serum T(3)/rT(3)
ratio correlated positively with liver D1 activity (P < 0.001) and
negatively with liver D3 activity (ns). These parameters were independent
of the type of insulin treatment. Liver D1 and serum T(3)/rT(3) were
highest in patients who died from severe brain damage, intermediate in
those who died from sepsis or excessive inflammation
Characterization of iodothyronine sulfatase activities in human and rat liver and placenta
In conditions associated with high serum iodothyronine sulfate
concentrations, e.g. during fetal development, desulfation of these
conjugates may be important in the regulation of thyroid hormone
homeostasis. However, little is known about which sulfatases are involved
in this process. Therefore, we investigated the hydrolysis of
iodothyronine sulfates by homogenates of V79 cells expressing the human
arylsulfatases A (ARSA), B (ARSB), or C (ARSC; steroid sulfatase), as well
as tissue fractions of human and rat liver and placenta. We found that
only the microsomal fraction from liver and placenta hydrolyzed
iodothyronine sulfates. Among the recombinant enzymes only the endoplasmic
reticulum-associated ARSC showed activity toward iodothyronine sulfates;
the soluble lysosomal ARSA and ARSB were inactive. Recombinant ARSC as
well as human placenta microsomes hydrolyzed iodothyronine sulfates with a
substrate preference for 3,3'-diiodothyronine sulfate (3,3'-T(2)S)
approximately T(3) sulfate (T(3)S) >> rT(3)S approximately T(4)S, whereas
human and rat liver microsomes showed a preference for 3,3'-T(2)S > T(3)S
>> rT(3)S approximately T(4)S. ARSC and the tissue microsomal sulfatases
were all characterized by high apparent K(m) values (>50 microM) for
3,3'-T(2)S and T(3)S. Iodothyronine sulfatase activity determined using
3,3'-T(2)S as a substrate was much higher in human liver microsomes than
in human placenta microsomes, although ARSC is expressed at higher levels
in human placenta than in human liver. The ratio of estrone sulfate to
T(2)S hydrolysis in human liver microsomes (0.2) differed largely from
that in ARSC homogenate (80) and human placenta microsomes (150). These
results suggest that ARSC accounts for the relatively low iodothyronine
sulfatase activity of human placenta, and that additional arylsulfatase(s)
contributes to the high iodothyronine sulfatase activity in human liver.
Further research is needed to identify these iodothyronine sulfatases, and
to study the physiological importance of the reversible sulfation of
iodothyronines in thyroid hormone metabolism
Effects of thyroid status and thyrostatic drugs on hepatic glucuronidation of lodothyronines and other substrates in rats - Induction of phenol UDP-glucuronyltransferase by methimazole
Glucuronidation of iodothyronines in rat liver is catalyzed by at least three UDP-glucuronyltransferases (UGTs): bilirubin UGT, phenol UGT, and androsterone UGT. Bilirubin and phenol UGT activities are regulated by thyroid hormone, but the effect of thyroid status on hepatic glucuronidation of iodothyronines is unknown. We examined the effects of hypothyroidism induced by treatment of rats with propylthiouracil (PTU) or methimazole (MMI) or by thyroidectomy as well as the effects of T4-induced hyperthyroidism on the hepatic UGT activities for T4, T3, bilirubin, p-nitrophenol (PNP), and androsterone. Bilirubin UGT activity was increased in MMI- or PTU-induced hypothyroid and thyroidectomized rats, and decreased in hyperthyroid animals. T4 and, to a lesser extent, T3 UGT activities were increased in MMI- or PTU-induced hypothyroid rats, and T4 but not T3 glucuronidation also showed a significant increase in thyroidectomized rats. T4 but not T3 UGT activity was slightly decreased in hyperthyroid rats. While PNP UGT activity was decreased in thyroidectomized rats and increased in hyperthyroid animals, it was also markedly increased by MMI and slightly increased by PTU-induced hypothyroidism. In T4-substituted rats, MMI did not affect T4, T3, bilirubin and androsterone UGT activities but again strongly induced PNP UGT activity, indicating that this represented a direct induction of PNP UGT by the drug independent of its thyrostatic action. Androsterone UGT activity was hardly affected by thyroid status. Our results suggest a modest, negative control of the hepatic glucuronidation of thyroid hormone by thyroid status, which may be mediated by changes in bilirubin UGT activity. To our knowledge, this is the first report of the marked induction of a hepatic enzyme by MMI, which is not mediated by its thyroid hormone-lowering effect
Characterization of human iodothyronine sulfotransferases
Sulfation is an important pathway of thyroid hormone metabolism that
facilitates the degradation of the hormone by the type I iodothyronine
deiodinase, but little is known about which human sulfotransferase
isoenzymes are involved. We have investigated the sulfation of the
prohormone T4, the active hormone T3, and the metabolites rT3 and
3,3'-diiodothyronine (3,3'-T2) by human liver and kidney cytosol as well
as by recombinant human SULT1A1 and SULT1A3, previously known as
phenol-preferring and monoamine-preferring phenol sulfotransferase,
respectively. In all cases, the substrate preference was 3,3'-T2 >> rT3 >
T3 > T4. The apparent Km values of 3,3'-T2 and T3 [at 50 micromol/L
3'-phosphoadenosine-5'-phosphosulfate (PAPS)] were 1.02 and 54.9
micromol/L for liver cytosol, 0.64 and 27.8 micromol/L for kidney cytosol,
0.14 and 29.1 micromol/L for SULT1A1, and 33 and 112 micromol/L for
SULT1A3, respectively. The apparent Km of PAPS (at 0.1 micromol/L 3,3'-T2)
was 6.0 micromol/L for liver cytosol, 9.0 micromol/L for kidney cytosol,
0.65 micromol/L for SULT1A1, and 2.7 micromol/L for SULT1A3. The sulfation
of 3,3'-T2 was inhibited by the other iodothyronines in a
concentration-dependent manner. The inhibition profiles of the 3,3'-T2
sulfotransferase activities of liver and kidney cytosol obtained by
addition of 10 micromol/L of the various analogs were better correlated
with the inhibition profile of SULT1A1 than with that of SULT1A3. These
results indicate similar substrate specificities for iodothyronine
sulfation by native human liver and kidney sulfotransferases and
recombinant SULT1A1 and SULT1A3. Of the latter, SULT1A1 clearly shows the
highest affinity for both iodothyronines and PAPS, but it remains to be
established whether it is the prominent isoenzyme for sulfation of thyroid
hormone in human liver and kidney
Different effects of continuous infusion of interleukin-1 and interleukin-6 on the hypothalamic-hypophysial-thyroid axis
The cytokines interleukin-1 (IL-1) and IL-6 are thought to be important
mediators in the suppression of thyroid function during nonthyroidal
illness. In this study we compared the effects of IL-1 and IL-6 infusion
on the hypothalamus-pituitary-thyroid axis in rats. Cytokines were
administered by continuous ip infusion of 4 micrograms IL-1 alpha/day for
1, 2, or 7 days or of 15 micrograms IL-6/day for 7 days. Body weight and
temperature, food and water intake, and plasma TSH, T4, free T4 (FT4), T3,
and corticosterone levels were measured daily, and hypothalamic pro-TRH
messenger RNA (mRNA) and hypophysial TSH beta mRNA were determined after
termination of the experiments. Compared with saline-treated controls,
infusion of IL-1, but not of IL-6, produced a transient decrease in food
and water intake, a transient increase in body temperature, and a
prolonged decrease in body weight. Both cytokines caused transient
decreases in plasma TSH and T4, which were greater and more prolonged with
IL-1 than with IL-6, whereas they effected similar transient increases in
the plasma FT4 fraction. Infusion with IL-1, but not IL-6, also induced
transient decreases in plasma FT4 and T3 and a transient increase in
plasma corticosterone. Hypothalamic pro-TRH mRNA was significantly
decreased (-73%) after 7 days, but not after 1 or 2 days, of IL-1 infusion
and was unaffected by IL-6 infusion. Hypophysial TSH beta mRNA was
significantly decreased after 2 (-62%) and 7 (-62%) days, but not after 1
day, of IL-1 infusion and was unaffected by IL-6 infusion. These results
are in agreement with previous findings that IL-1, more so than IL-6,
directly inhibits thyroid hormone production. They also indicate that IL-1
and IL-6 both decrease plasma T4 binding. Furthermore, both cytokines
induce an acute and dramatic decrease in plasma TSH before (IL-1) or even
without (IL-6) a decrease in hypothalamic pro-TRH mRNA or hypophysial TSH
beta mRNA, suggesting that the acute decrease in TSH secretion is not
caused by decreased pro-TRH and TSH beta gene expression. The
TSH-suppressive effect of IL-6, either administered as such or induced by
IL-1 infusion, may be due to a direct effect on the thyrotroph, whereas
additional effects of IL-1 may involve changes in the hypothalamic release
of somatostatin or TRH.(ABSTRACT TRUNCATED AT 400 WORDS