20 research outputs found
Thyroid hormone uptake in cultured rat anterior pituitary cells: effects of energy status and bilirubin
Transport of thyroxine (T(4)) into the liver is inhibited in fasting and
by bilirubin, a compound often accumulating in the serum of critically ill
patients. We tested the effects of chronic and acute energy deprivation,
bilirubin and its precursor biliverdin on the 15-min uptake of
[(125)I]tri-iodothyronine ([(125)I]T(3)) and [(125)I]T(4) and on TSH
release in rat anterior pituitary cells maintained in primary culture for
3 days. When cells were cultured and incubated in medium without glucose
and glutamine to induce chronic energy deprivation, the ATP content was
reduced by 45% (P<0. 05) and [(125)I]T(3) uptake by 13% (NS), but TSH
release was unaltered. Preincubation (30 min) and incubation (15 min) with
10 microM oligomycin reduced ATP content by 51% (P<0.05) and 53% (P<0. 05)
under energy-rich and energy-poor culture conditions respectively;
[(125)I]T(3) uptake was reduced by 66% (P<0.05) and 64% (P<0.05). Neither
bilirubin nor biliverdin (both 1-200 microM) affected uptake of
[(125)I]T(3) or [(125)I]T(4). Bilirubin (1-50 microM) did not alter basal
or TRH-induced TSH release. In conclusion, the absence of inhibitory
effects of chronic energy deprivation and bilirubin on thyroid hormone
uptake by pituitary cells supports the view that the transport is
regulated differently than that in the liver
Uptake of triiodothyronine and triiodothyroacetic acid in neonatal rat cardiomyocytes: effects of metabolites and analogs
Cellular and nuclear uptake of [125I]tri-iodothyronine (T3) and
[125I]triiodothyroacetic acid (Triac) were compared in cardiomyocytes of
2-3 day old rats, and the effect of thyroid hormone analogs on cellular
T(3) uptake was measured. Cells (5-10 x 10(5) per well) were cultured in
DMEM-M199 with 5% horse serum and 5% FCS. Incubations were performed for
from 15 min to 24 h at 37 degrees C in the same medium, 0.5% BSA and
[125I]T3 (100 pM), or [125I]Triac (240 pM). Expressed as % dose, T(3)
uptake was five times Triac uptake, but expressed as fmol/pM free hormone,
Triac uptake was at least 30% (P<0.001) greater than T3 uptake, whereas
the relative nuclear binding of the two tracers was comparable. The 15 min
uptake of [125I]T3 was competitively inhibited by 10 microM unlabeled T3
(45-52%; P<0.001) or 3,3'- diiodothyronine (T2) (52%; P<0.001), and to a
smaller extent by thyroxine (T(4)) (27%; 0.05<P<0.1). In contrast, 10
microM 3,5-T2, Triac, or tetraiodothyroacetic acid (Tetrac) did not affect
T3 uptake after 15 min or after 24 h. Diiodothyropropionic acid (DITPA)
(10 microM) reduced 15-min T3 uptake by about 24% (P<0.05), but it had a
greater effect after 4 h (56%; P<0.001). Exposure to 10 nM DITPA during
culture reduced cellular T3 uptake, as did 10 nM T3, suggesting
down-regulation of the plasma membrane T3 transporters. We conclude that
i) Triac is taken up by cardiomyocytes; ii) 3,3'-T2 and, to a lesser
extent, DITPA and T4 interfere with plasma membrane transport of T3,
whereas 3,5-T2, Triac, or Tetrac do not; iii) the transport mechanism for
Triac is probably different from that for T3
Adaptive changes in transmembrane transport and metabolism of triiodothyronine in perfused livers of fed and fasted hypothyroid and hyperthyroid rats
The transport and subsequent metabolism of triiodothyronine (T3) were studied in isolated perfused livers of euthyroid, hypothyroid, and hyperthyroid rats, both fed and 48-hour-fasted. T3 kinetics (transport and metabolism) during perfusion were evaluated by a two-pool model, whereas the metabolism of T3 was also investigated by determination of T3 breakdown products by chromatography of medium and bile. For comparison of groups, metabolism was corrected for differences in transport. Transport parameters in fed hypothyroid livers were not significantly changed as compared with euthyroid livers, whereas metabolism was decreased. In fed hyperthyroid livers, fractional transfer rate constants for influx (k21) and efflux (k12) were decreased and metabolism, corrected for differences in intracellular mass transfer, was increased. Furthermore, for transport in hyperthyroid livers it was shown that only total mass transfer (TMT) into the metabolizing liver compartment (not into the nonmetabolizing liver compartment) was decreased. Transport and metabolic parameters in fasted hypothyroid livers were decreased as compared with euthyroid fed livers. In fasted hyperthyroid livers, transport and metabolism were not significantly different as compared with that in euthyroid fed livers, so transport was increased versus hyperthyroid fed livers. It appeared therefore that fasting normalized the effects of hyperthyroidism on both the transport and metabolic processes of T3 in the liver. The present study demonstrates normal transport and decreased metabolism in livers of hypothyroid fed rats and decreased transport and increased metabolism in livers of hyperthyroid fed rats. In livers of hypothyroid fasted rats transport and metabolism were decreased, whereas in livers of hyperthyroid fasted rats transport and metabolism were not significantly different from that in euthyroid fed livers. These changes might favor tissue euthyroidism despite the altered thyroid and nutritional state, and can therefore be seen as adaptation mechanisms to these altered states at the tissue level
Rapid sulfation of 3,3',5'-triiodothyronine in native Xenopus laevis oocytes
Sulfation is an important metabolic pathway facilitating the degradation
of thyroid hormone by the type I iodothyronine deiodinase. Different human
and rat tissues contain cytoplasmic sulfotransferases that show a
substrate preference for 3,3'-diiodothyronine (3,3'-T2) > T3 > rT3 > T4.
During investigation of the expression of plasma membrane transporters for
thyroid hormone by injection of rat liver RNA in Xenopus laevis oocytes,
we found uptake and metabolism of iodothyronines by native oocytes. Groups
of 10 oocytes were incubated for 20 h at 18 C in 0.1 ml medium containing
500,000 cpm (1-5 nM) [125I]T4, [125I]T3, [125I]rT3, or [125I]3,3'-T2. In
addition, cytosol prepared from oocytes was tested for iodothyronine
sulfotransferase activity by incubation of 1 mg cytosolic protein/ml for
30 min at 21 C with 1 microM [125I]T4, [125I]T3, [125I]rT3, or
[125I]3,3'-T2 and 50 microM 3'-phosphoadenosine-5'-phosphosulfate.
Incubation media, oocyte extracts, and assay mixtures were analyzed by
Sephadex LH-20 chromatography for production of conjugates and iodide.
After 20-h incubation, the percentage of added radioactivity present as
conjugates in the media and oocytes amounted to 0.9 +/- 0.2 and 1.0 +/-
0.1 for T4, less than 0.1 and less than 0.1 for T3, 32.5 +/- 0.4 and 29.3
+/- 0.2 for rT3, and 3.8 +/- 0.3 and 2.3 +/- 0.2 for 3,3'-T2, respectively
(mean +/- SEM; n = 3). The conjugate produced from rT3 was identified as
rT3 sulfate, as it was hydrolyzed by acid treatment. After injection of
oocytes with copy RNA coding for rat type I iodothyronine deiodinase, we
found an increase in iodide production from rT3 from 2.3% (water-injected
oocytes) to 46.2% accompanied by a reciprocal decrease in rT3 sulfate
accumulation from 53.7% to 7.1%. After 30-min incubation with cytosol and
3'-phosphoadenosine-5'-phosphosulfate, sulfate formation amounted to 1.8%
for T4, less than 0.1% for T3, 77.9% for rT3, and 2.9% for 3,3'-T2. These
results show that rT3 is rapidly metabolized in native oocytes by
sulfation. The substrate preference of the sulfotransferase activity in
oocytes is rT3 >> 3,3'-T2 > T4 > T3. The physiological significance of the
high activity for rT3 sulfation in X. laevis oocytes remains to be
established
Changes in renal tri-iodothyronine and thyroxine handling during fasting
OBJECTIVE: Liver handling of thyroid hormones (TH) has been known to alter
significantly during fasting. This study investigates whether renal
handling of TH is also changed during fasting. METHODS: We measured
urinary excretion rates and clearances of free tri-iodothyronine (T(3))
and free thyroxine (T(4)) in healthy subjects prior to and on the third
day of fasting. RESULTS: During fasting, both mean T(3) and T(4) urinary
excretion decreased significantly to a mean value of 42% of control. Also,
total and free (F) serum T(3) concentrations declined significantly, but
serum T(4) did not change. Both FT(3) and FT(4) clearance decreased
significantly during fasting (62% and 42% of control). The fasting-induced
decrease in uric acid clearance correlated well with the decrease in FT(3)
clearance (r=0.94; P<0.001). Serum concentrations of non-esterified fatty
acids (NEFA) were significantly elevated during fasting. CONCLUSIONS: The
findings cannot be fully explained by the fasting-induced decrease in
serum T(3), a
Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability
Although it was originally believed that thyroid hormones enter target
cells by passive diffusion, it is now clear that cellular uptake is
effected by carrier-mediated processes. Two stereospecific binding sites
for each T4 and T3 have been detected in c
Effects of interleukin-1 beta on thyrotropin secretion and thyroid hormone uptake in cultured rat anterior pituitary cells
The effects of interleukin-1 beta (IL-1 beta) and tumor necrosis
factor-alpha (TNF alpha) on basal and TRH-induced TSH release, and the
effects of IL-1 beta on the uptake of [125I]T3 and [125I]T4 and on nuclear
binding of [125I]T3 were examined. Furthermore, the release of other
anterior pituitary hormones in the presence of IL-1 beta was measured.
Anterior pituitary cells from male Wistar rats were cultured for 3 days in
medium containing 10% FCS. Incubation were performed at 37 C in medium
with 0.5% BSA for measurement of [125I]T3 uptake and with 0.1% BSA for
measurement of [125I]T4 uptake. Exposure to IL-1 beta (1 pM-1 nM) or TNF
alpha (100 pM) for 2-4 h resulted in a significant decline in TSH release,
which was almost 50% (P < 0.05) for 1 nM IL-1 beta and 24% (P < 0.05) for
100 pM TNF alpha. Measurement of other anterior pituitary hormones (FSH,
LH, PRL, and ACTH) in the same incubation medium showed that IL-1 beta did
not alter their release. When the effects of IL-1 beta (1 pM-1 nM) and TNF
alpha (100 pM) on TRH-induced TSH release were measured in short term
experiments, the inhibitory effects had disappeared. The addition of 1-100
nM octreotide, a somatostatin analog, resulted in a decrease in
TRH-induced TSH release up to 33% of the control value (P < 0.05).
Exposure to dexamethasone (1 nM to 1 microM) affected basal and
TRH-induced TSH release similar to the effect of IL-1 beta. The 15-min
uptake of [125I]T3 and [125I]T4, expressed as femtomoles per pM free
hormone, was not affected by the presence of IL-1 beta (1-100 pM). When
IL-1 beta (100 pM) was present during 3 days of culture, TSH release was
reduced to 88 +/- 2% of the control value (P < 0.05). This effect was not
associated with an altered [125I]T3 uptake (15 min to 4 h) or with any
change in nuclear T3 binding. We conclude that 1) IL-1 beta decreases TSH
release by a direct action on the pituitary; 2) this effect is not due to
elevated thyroid hormone uptake or increase T3 nuclear occupancy; 3) IL-1
beta does not affect TRH-induced TSH release or the release of other
anterior pituitary hormones; and 4) TNF alpha affects basal and
TRH-induced TSH release in the same way as IL-1 beta
Thyroid hormone transport by the heterodimeric human system L amino acid transporter
Transport of thyroid hormone across the cell membrane is required for
thyroid hormone action and metabolism. We have investigated the possible
transport of iodothyronines by the human system L amino acid transporter,
a protein consisting of the human 4F2 heavy chain and the human LAT1 light
chain. Xenopus oocytes were injected with the cRNAs coding for human 4F2
heavy chain and/or human LAT1 light chain, and after 2 d were incubated at
25 C with 0.01-10 microM [(125)I]T(4), [(125)I]T(3), [(125)I]rT(3), or
[(125)I]3,3'-diiodothyronine or with 10-100 microM [(3)H]arginine,
[(3)H]leucine, [(3)H]phenylalanine, [(3)H]tyrosine, or [(3)H]tryptophan.
Injection of human 4F2 heavy chain cRNA alone stimulated the uptake of
leucine and arginine due to dimerization of human 4F2 heavy chain with an
endogenous Xenopus light chain, but did not affect the uptake of other
ligands. Injection of human LAT1 light chain cRNA alone did not stimulate
the uptake of any ligand. Coinjection of cRNAs for human 4F2 heavy chain
and human LAT1 light chain stimulated the uptake of phenylalanine >
tyrosine > leucine > tryptophan (100 microM) and of 3,3'-diiodothyronine >
rT(3) approximately T(3) > T(4) (10 nM), which in all cases was Na(+)
independent. Saturation analysis provided apparent Michaelis constant
(K(m)) values of 7.9 microM for T(4), 0.8 microM for T(3), 12.5 microM for
rT(3), 7.9 microM for 3,3'-diiodothyronine, 46 microM for leucine, and 19
microM for tryptophan. Uptake of leucine, tyrosine, and tryptophan (10
microM) was inhibited by the different iodothyronines (10 microM), in
particular T(3). Vice versa, uptake of 0.1 microM T(3) was almost
completely blocked by coincubation with 100 microM leucine, tryptophan,
tyrosine, or phenylalanine. Our results demonstrate stereospecific
Na(+)-independent transport of iodothyronines by the human heterodimeric
system L amino acid transporter
Expression of rat liver cell membrane transporters for thyroid hormone in Xenopus laevis oocytes
The present study was conducted to explore the possible use of Xenopus
laevis oocytes for the expression cloning of cell membrane transporters
for iodothyronines. Injection of stage V-VI X. laevis oocytes with 23 ng
Wistar rat liver polyadenylated RNA (mRNA) resulted after 3-4 days in a
highly significant increase in [125I]T3 (5 nM) uptake from 6.4 +/- 0.8
fmol/oocyte x h in water-injected oocytes to 9.2 +/- 0.65 fmol/oocyte x h
(mean +/- SEM; n = 19). In contrast, [125I]T4 (4 nM) uptake was not
significantly stimulated by injection of total liver mRNA. T3 uptake
induced by liver mRNA was significantly inhibited by replacement of Na+ in
the incubation medium by choline+ or by simultaneous incubation with 1
microM unlabeled T3. In contrast, T3 uptake by water-injected oocytes was
not Na+ dependent. Fractionation of liver mRNA on a 6-20% sucrose gradient
showed that maximal stimulation of T3 uptake was obtained with mRNA of
0.8-2.1 kilobases (kb). In contrast to unfractionated mRNA, the 0.7- to
2.1-kb fraction also significantly stimulated transport of T4, and it was
found to induce uptake of T3 sulfate (T3S). Because T3S is a good
substrate for type I deiodinase (D1), 2.3 ng rat D1 complementary RNA
(cRNA) were injected either alone or together with 23 ng of the 0.8- to
2.1-kb fraction of rat liver mRNA. Compared with water-injected oocytes,
injection of D1 cRNA alone did not stimulate uptake of [125I]T3S (1.25
nM). T3S uptake in liver mRNA and D1 cRNA-injected oocytes was similar to
that in oocytes injected with mRNA alone, showing that transport of T3S is
independent of the metabolic capacity of the oocyte. Furthermore,
coinjection of liver mRNA and D1 cRNA strongly increased the production of
125I-, showing that the T3S taken up by the oocyte is indeed transported
to the cell interior. In conclusion, injection of rat liver mRNA into X.
laevis oocytes resulted in a stimulation of saturable, Na+-dependent T4,
T3 and T3S transport, indicating that rat liver contains mRNA(s) coding
for plasma membrane transporters for these iodothyronine derivatives
Uptake of triiodothyronine sulfate and suppression of thyrotropin secretion in cultured anterior pituitary cells
To investigate the uptake of triiodothyronine sulfate (T3S) and its effect on thyrotropin-releasing hormone (TRH)-induced thyrotropin (TSH) secretion, anterior pituitary cells were isolated from euthyroid rats and cultured for 3 days in medium containing 10% fetal calf serum. Incubation was performed at 37°C in medium containing 0.5% bovine serum albumin (BSA). Exposure of the pituitary cells to TRH (0.1 μmol/L) for 2 hours stimulated TSH secretion by 176%. This effect was reduced by approximately 45% after a 2-hour preincubation with T3 (0.001 to 1 μmol/L). A significant inhibitory effect of T3S on TRH-induced TSH release was only observed at a concentration of 1 μmol/L. The uptake of [125I]T3 after 1 hour of incubation was reduced by 40% ± 4% (P < .001) by simultaneous addition of 10 nmol/L unlabeled T3, whereas 1 μmol/L T3S was required to obtain a reduction of the [125I]T3 uptake by 34% ± 2% (P < .001). The amount of T3 present in the unlabeled T3S preparation was 0.25% as determined by radioimmunoassay. When pituitary cells were incubated for 1 hour with [125I]T3S or [125I]T3 both 50,000 cpm/0.25 mL), the uptake of [125I]T3zS expressed as a percentage of the dose was 0.04% ± 0.02% (mean ± SE, n = 4), whereas that of [125I]T3 amounted to 3.0% ± 0.4% (n = 4). In contrast, when hepatocytes were incubated for 1 hour with [125I]T3S, the uptake amounted to 5.1% ± 0.8% (n = 9), whereas that of [125I]T3 was 22.1% ± 1.7% (n = 9). Furthermore, [125I]T3S was as rapidly deiodinated (iodide production, 14.9% ± 2.6%; n = 9) as [125I]T3 (12.1% ± 0.8%, n = 9) by hepatocytes. It is concluded that (1) T3S is poorly taken up by pituitary cells, and (2) the suppressive effect of high concentrations of T3S on TRH-induced TSH secretion and on [125I]T3 uptake can be explained by slight contamination with T3. Thus, it appears that T3S has only a minor biological effect, if any, on the pituitary