Splanchnic extraction of 3,3'-diiodothyronine and 3',5'-diiodothyronine in hyperthyroidism

The splanchnic extraction of 3,3'-diiodothyronine (3,3'-T2) and 3',5'-diiodothyronine (3',5'-T2) was studied in 7 hyperthyroid patients and 20 normal subjects employing the hepatic venous catheterization technique. A significant net uptake by splanchnic tissues was found for both diiodothyronines . The fractional splanchnic extraction calculated as the arterio-hepatic venous plasma concentration difference divided by the arterial concentration was unaffected by hyperthyroidism as compared to normal values. There was a close positive correlation between the arterio-hepatic venous concentration difference and arterial concentration, 3,3'-T2: r = 0.988, and 3',5'-T2: r = 0.932 (P less than 0.001). The splanchnic extraction was nonsaturable at endogenous plasma concentrations of 3,3'-T2 up to at least 17.0 ng/dl and of 3',5'-T2 up to at least 15.2 ng/dl. The data suggest that the splanchnic extraction of 3,3'-T2 and 3',5'-T2 obeys first order kinetics, the fractional extraction being unaffected by hyperthyroidism. Furthermore, changes in the net splanchnic extraction of 3,3'-T2 and 3',5'-T2 do not seem to contribute to changes in circulating levels of these iodothyronines. It is suggested that tissues other than the liver contribute significantly to the deiodination process both in normal and in hyperthyroid man.     

The metabolism of 3,3'-diiodothyronine in man

The production rate of 3,3'-Diiodothyronine (3,3'-T2) was measured in five healthy subjects after a single injection of [125I]3,3'-T2. The [125I]3,3'-T2 was measured by immunoprecipitation. To reduce the large amount of nonspecific serum radioactivity (iodides, 3,3'-T2 metabolites, and protein-bound iodine), the sear were treated before the immunoprecipitation with an anion exchanger and polyethylene glycol (final concentration, 10%). The noncompartmental analysis of the data gave the following results: MCR, 0.52 +/- 0.07 liters/min or 926 +/- 142 liters/day (mean +/- SD); and production rate, 23.7 +/- 8.2 ng/min or 34 +/- 12 micrograms/day.     

Effect of 3,3'-diiodothyronine and 3,5-diiodothyronine on rat liver oxidative capacity.

We report that 3,5,3'-triiodothyronine (T3) as well as two other iodothyronines (3,3'-diiodothyronine and 3,5-diiodothyronine (T2s)) stimulate rat liver oxidative capacity (measured as cytochrome oxidase activity (COX)). In hypothyroid rats COX activity and mitochondrial protein content are significantly lower than in normal control animals. The administration of both T3 and T2s to hypothyroid rats significantly enhances hepatic COX activity with T3 having the greatest effect (+60%); moreover, T3 restores the mitochondrial protein content whereas the T2s are ineffective. Administration of T2s results in a faster stimulation (already significant 1 h after the injection) of hepatic COX activity than T3 injection. Our results suggest that T3 acts on the protein synthesis mechanism involved in the regulation of the mitochondrial mass while T2s would act directly at the mitochondrial level.     

Metabolism of triiodothyronine in rat hepatocytes

The metabolism of T3 by isolated rat hepatocytes was analyzed by Sephadex LH-20 chromatography, HPLC, and RIA for T3 sulfate (T3S) and 3,3'-diiodothyronine (3,3'-T2). Type I iodothyronine deiodinase activity was inhibited with propylthiouracil (PTU), and phenol sulfotransferase activity by SO4(2-) depletion or with competitive substrates or inhibitors. Under normal conditions, labeled T3 glucuronide and I- were the main products of [3'-125I]T3 metabolism. Iodide production was decreased by inhibition (PTU) or saturation (greater than 100 nM T3) of type I deiodinase, which was accompanied by the accumulation of T3S and 3,3'-T2S. Inhibition of phenol sulfotransferase resulted in decreased iodide production, which was associated with an accumulation of 3,3'-T2 and 3,3'-T2 glucuronide, independent of PTU. Formation of 3,3'-T2 and its conjugates was only observed at T3 substrate concentrations below 10 nM. Thus, T3 is metabolized in rat liver cells by three quantitatively important pathways: glucuronidation, sulfation, and direct inner ring deiodination. Whereas T3 glucuronide is not further metabolized in the cultures, T3S is rapidly deiodinated by the type I enzyme. As confirmed by incubations with isolated rat liver microsomes, direct inner ring deiodination of T3 is largely mediated by a low Km, PTU-insensitive, type III-like iodothyronine deiodinase, and production of 3,3'-T2 is only observed if its rapid sulfation is prevented.     

Failure of 3,3'-diiodothyronine administration to alter TSH and prolactin responses to TRH stimulation.

In order to determine whether elevations in serum 3,3'-diiodothyronine (3,3'T2) concentrations influence the hypothalamic-pituitary--thyroid axis, thyrotropin (TSH) and prolactin responses to thyrotropin-releasing hormone (TRH) were assessed in five patients both prior to and during 3,3'T2 administration. Mean (+/- SE) peak TSH responses to TRH were 168 +/- 64 microU/ml during 3,3'T2 administration and 168 +/- 65 muU/ml during 3,3'T2 administration. Mean basal and peak prolactin concentrations after TRH were 6 +/- 3 ng/ml and 54 +/- 26 ng/ml, whereas during 3,3'T2 administration the basal and peak prolactin levels were 6 +/- 2 ng/ml and 55 +/- 28 ng/ml, respectively. Hypothyroid rats administered triiodothyronine (10 migrogram b.i.d.) for 5 days had a mean TSH response to TRH stimulation of 0.051 +/- 0.003 mU/ml, whereas rats to whom saline or 3,3'T2 (50 microgram b.i.d.) had been given for the same time interval had mean TRH-induced TSH responses of 1.127 +/- 0.179 mU/ml and 1.324 +/- 0.286 mU/ml, respectively. None of the TSH or prolactin responses to TRH, in either human or rat studies, were apparently altered by 3,3'T2. These observations suggest that elevation of serum 3,3'T2 levels are not associated with alterations in the hypothalamic--pituitary--thyroid axis in the experimental systems employed.     

Inhibition of uptake of thyroid hormone into rat hepatocytes by preincubation with N-bromoacetyl-3,3',5-triiodothyronine

To investigate whether affinity coupling of N-bromoacetyl-T3 (BrAcT3) to the T3 membrane carrier results in an inhibition of transport of T3 into the cell, rat hepatocytes in monolayer were incubated for 2 h at 21 C with 1.3 mumol/liter BrAcT3 in medium without protein. After extensive washing, cells were incubated during 20 h at 37 C with [125I]T3 in medium with 0.5% BSA, and products in supernatants were analyzed by LH-20 column chromatography. In addition the apparent affinity constant (Km) and maximal uptake velocity (Vmax) of the high affinity uptake process were estimated using 1 min incubations of hepatocytes with various concentrations of T3. In control experiments (i.e. without BrAcT3 affinity coupling) about 57% of the added T3 was cleared from the medium and further metabolized, 85% of the cleared T3 reappeared in the medium as I-, 15% as conjugates. Addition of propylthiouracil during the 20 h incubation with T3 strongly inhibited deiodination, without a change in T3 clearance. Because T3 is sulfated before deiodination, a concomitant rise in conjugates was observed. Addition of ouabain to control cells during the 20 h incubation with T3 strongly inhibited uptake, with a parallel decrease in I- and conjugate formation. After affinity coupling of BrAcT3, T3 clearance was inhibited (by 30% P less than 0.001). Since I- production was more depressed (by 73%) than T3 clearance, with some rise in conjugate formation (P less than 0.001), inhibition of deiodinase by BrAcT3 also took place. The effects of BrAcT3 and ouabain on uptake of T3 appeared to be additive as were the effects of propylthiouracil and BrAcT3 on deiodination. After affinity coupling of BrAcT3, the Km of T3 uptake did not change significantly; however Vmax was 54% lower (P less than 0.025) indicating a noncompetitive inhibition of the transport system. Preincubation of the cells with N-acetyl-T3 does not alter the characteristics of uptake of T3 by rat hepatocytes as compared to controls, indicating that no binding of this compound occurs. It is concluded that preincubation of hepatocytes with BrAcT3 diminished I- formation from T3; 50% of this inhibition is due to decreased membrane transport and 50% by reduction of deiodination. Inhibition of membrane transport by BrAcT3 is substantiated by a 54% lower Vmax without a significant change in Km as compared to control. The effect of transport of thyroid hormone on metabolism stresses the importance of the membrane carrier in the translocation process.     

Renal handling of thyroxine, 3,5,3'- and 3,3',5'-triiodothyronine, 3,3'- and 3',5'-diiodothyronine in man

The 24-h urinary excretion and renal clearance of thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), and 3',5'-diiodothyronine (3',5'-T2) were measured in 17 healthy subjects. The median urinary excretion was (pmol/24h) T4: 1242, T3: 828, rT3: 12.9, 3,3'-T2: 331, and 3',5'-T2: 5.8. The corresponding renal clearances were in median (ml/min) T4: 31, T3: 133, rT3: 15, 3,3'-T2: 683, and 3',5'-T2: 4.5. The clearances differed mutually (P less than 0.01) as well as from the creatinine clearance (P less than 0.01) which was in median 87 ml/min. Thus, all iodothyronines studied were subject to tubular transport mechanisms besides glomerular filtration. The 3 iodothyronines with 2 iodine atoms in the phenolic ring of the thyronine molecule, T4, rT3 and 3',5'-T2, were mainly tubularly reabsorbed, whereas those with only one iodine atom in the phenolic ring, T3 and 3,3'-T2, were mainly tubularly secreted. It might be hypothesized that the number of iodine atoms in the phenolic ring determines the direction of the tubular transport (presence of 2 iodine atoms is associated with tubular reabsorption, and of one iodine atom with secretion), whereas the rate of tubular transport decreases with decreasing number of iodine atoms in the tyrosylic ring.     

Metabolism of thyroid hormones in isolated rat hepatocytes: studies on the influences of carbamazepine and phenytoin

The in vitro handling of thyroid hormones was studied in isolated rat hepatocytes by measuring 1) the cellular uptake of T4, 2) the conversion of T4 to T3 and 3) the degradation of T4 and T3. The in vitro conversion of T4 to T3 increased significantly by adding ethanol 2% or carbamazepine (CBZ) 400 microM in ethanol 2% to the incubation medium. As there was no difference between ethanol and CBZ/ethanol on the T3 formation, this effect was probably caused by ethanol. The T3 formation was unaffected by phenytoin (PHT) in conc. up to 400 microM, while propylthiouracil (PTU) 100 and 400 microM inhibited the conversion completely. The T4 to T3 conversion in hepatocytes from rats pretreated with CBZ or PHT for 2 weeks was not significantly different from untreated controls. The cellular uptake of T4 was reduced by about 30% in the presence of PHT and unaltered by CBZ and ethanol. The degradation of T4 and T3 was not influenced by the in vitro addition of CBZ or PHT, nor was the degradation of T4 and T3 significantly different from untreated controls in hepatocyte suspensions from CBZ or PHT pretreated rats. Our findings suggest that the handling of thyroid hormones in isolated rat hepatocytes is not influenced by the in vitro or in vivo exposure to CBZ or PHT.     

A radioimmunoassay for measurement of 3,3'-diiodothyronine sulfate: Studies in thyroidal and nonthyroidal diseases, pregnancy, and fetal/neonatal life

Data suggesting that (1) sulfation of the phenolic hydroxyl of iodothyronines plays an important role in thyroid hormone metabolism and (2) maternal serum 3,3'-diiodothyronone sulfate (3,3'-T(2)S) may reflect on the status of fetal thyroid function stimulated us to develop a radioimmunoassay (RIA) for measurement of T(2)S. Our T(2)S RIA is highly sensitive, practical, and reproducible. T(4)S, T(3)S, and T(1)S crossreacted 3.1%, 0.81%, and 5.3%, respectively; thyroxine (T(4)), triiodothyronine (T(3)), and reverse (r)T(3), 3,3'-T(2) and 3'-T(1) crossreacted <0.1%. Although rT(3) sulfate (rT(3)S) crossreacted 55% in 3,3'-T(2)S RIA, its serum levels are very low and have little influence on serum T(2)S values reported here. T(2)S was measured in ethanol extracts of serum, amniotic fluid, and urine. Recovery of nonradioactive T(2)S added to serum was 96%. The dose-response curves of inhibition of binding of (125)I-T(2)S to anti-T(2)S by serial dilutions of ethanol extracts of serum or urine were essentially parallel to the standard curve. The detection threshold of the RIA varied between 0.17 and 0.50 nmol/L (or 10 and 30 ng/dL). The coefficient of variation (CV) averaged 9% within an assay and 13% between assays. The serum concentration of T(2)S was [mean +/- SE, nmol/L] 0.86 +/- 0.59 in 36 normal subjects, 2.2 +/- 0.06 in 10 hyperthyroid patients (P <.05), 0.73 +/- 0.10 in 11 hypothyroid patients (not significant [NS]), 6.0 +/- 1.5 in 16 patients with systemic nonthyroidal illness (P <.001), 18 +/- 2.5 in 16 newborn cord blood sera (P <.02), 2.7 +/- 0.32 in 25 pregnant women [15 to 40 weeks gestation, P <.001], 0.94 +/- 0.10 in 10 hypothyroid women receiving T(4) replacement therapy (NS), and 2.0 +/- 0.38 in 11 hypothyroid women treated with T(4) replacement and oral contraceptives (P <.02); serum T(2)S levels in the third trimester of pregnancy were similar to those in the second trimester of pregnancy. T(2)S concentration in amniotic fluid was 12.5 +/- 2.7 nmol/L (n = 7) at 15 to 20 weeks gestation, and it decreased markedly to 3.3 +/- 1.3 nmol/L (n = 3) at 35 to 38 weeks gestation. Urinary excretion of T(2)S in random urine samples of 19 normal subjects was 10.9 +/- 1.3 nmol/g creatinine. (1) T(2)S is a normal component of human serum, urine, and amniotic fluid, and serum T(2)S levels change substantially in several physiologic and pathologic conditions; (2) high serum T(2)S in pregnancy may signify increased transfer of T(2)S from fetal to maternal compartment, estrogen-induced increase in T(2)S production, decreased clearance, or a combination of these factors. The data do not support the notion that fetal thyroid function is the only or the predominant factor responsible for high serum T(2)S in pregnant women.     

Glutathione deficiency induced by cystine and/or methionine deprivation does not affect thyroid hormone deiodination in cultured rat hepatocytes and monkey hepatocarcinoma cells

To elucidate the recently advanced hypothesis that glutathione [L-gamma-glutamyl-L-cysteinyl glycine (GSH)] regulates deiodinating enzyme activities, accounting for the decreased conversion of T4 to T3 in the liver of fetal and starved animals, we investigated thyroid hormone metabolism in GSH-depleted neoplastic and normal hepatocytes. In monkey hepatocarcinoma cells, intracellular total GSH decreased below 10% of the control value (approximately 25 micrograms/mg protein) when cells were grown for 44 h in medium deficient in cystine and methionine or in cystine alone. The latter finding indicated that transsulfuration from methionine to cysteine was defective in these neoplastic cells. In primary cultured adult rat hepatocytes, on the other hand, the transsulfuration pathway was intact, and total GSH decreased below 10% of control (approximately 20 micrograms/mg protein) only in cells grown in cystine- and methionine-deficient medium. In both cell types, the oxidized GSH fraction remained constant (2-5% of total). Incubation with 125I-labeled T4 and T3, followed by chromatography, was used to evaluate 5-deiodination in hepatocarcinoma cells and both 5- and 5'-deiodination in normal hepatocytes. Deiodination was not decreased by GSH deficiency in either case, but was actually increased in hepatocarcinoma cells. This resulted from an increase in the Vmax of 5-deiodinase related to growth arrest. Diamide at 2 mM reversibly inhibited both 5'- and 5'-deiodination in rat hepatocytes, accompanied by decreased total GSH as well as increased GSH disulfide (27% of total). The data suggest that GSH is so abundant in the liver that hepatocytes can tolerate a greater than 90% decrease in intracellular concentration without any change in thyroid hormone deiodination and indicate that altered thyroid hormone metabolism in the fetus and in starvation cannot be accounted for by a decreased hepatic GSH concentration.     

Local interaction of apoptotic hepatocytes and Kupffer cells in a rat model of systemic endotoxemia

Aim: There is strong evidence that hepatocellular apoptosis is not only initiated by circulating blood cells which become adherent within the endotoxemic liver, but also contributes to further sustain the inflammatory cell-cell response. Methods: Because previous studies assumed the importance of the role of cellular cross-talk in mediating inflammatory liver injury, we herein examined the activation of Kupffer cells (KCs) and their spatial coincidence with intrahepatic leukocyte adherence and hepatocellular apoptosis at 6 h after intraperitoneal exposure of rats with lipopolysaccharide (10 mg/kg). Results: In vivo multifluorescence microscopy revealed liver injury including nutritive perfusion failure, tissue hypoxia, leukocyte accumulation, as well as KC activation and parenchymal apoptotic cell death. Detailed spatial analysis revealed frequent colocalization of activated KCs with apoptotic hepatocytes. Colocalization was absent in saline-treated controls.Colocalization was confirmed by histochemistry, which showed ED1-positive KCs neighboring and engulfing TUNEL-positive hepatocytes. Colocalization of KCs with leukocytes ranged between 4% and 5% and did not increase in endotoxemic animals. Taken together, the present results indicate that apoptotic cell death of hepatocytes may stimulate phagocytosis by neighboring KCs. Direct KC-leukocyte contact seems not to be mandatory for cellular communication in the process of hepatocellular apoptosis. Conclusion: With respect to the fundamental importance of cell apoptosis, improved knowledge of these cell-cell interactions might allow the development of new therapeutic strategies through the regulation of apoptotic cell death.     

Enzymatic sulfation of steroids: I. The enzymatic basis for the sex difference in cortisol sulfation by rat liver preparations.

Liver cytosols from female rats contained 6-8 times as much cortisol sulfotransferase activity as those from males. The reaction product, with both sexes, appeared to be cortisol-21 sulfate. Liver cytosols from male and female rats showed different substrate preferences when tested with cortisol, estradiol-17beta, testosterone, and dehydroepiandrosterone, suggesting that they contained different steroid sulfotransferases. Fractionation of cytosols from female rats on DEAE Sephadex A-50 columns resolved 3 steroid sulfotransferases, or families of steroid sulfotransferases (STI, STII, and STIII). These enzymes exhibited different substrate preferences. STI and STIII had the greatest preferences for cortisol, although none of the enzymes was restricted to the glucocorticoid. Fractionation of cytosols from males resolved 2 sulfotransferases which eluted at salt concentrations identical to STII and STIII from females. Study of the development of cortisol sulfotransferase activity with age showed little enzyme activity in rats of either sex at 2 days after birth. Enzyme activity developed in parallel in both sexes until 30 days after birth. Then the sulfotransferase activity began to rise in females and to drop in males. By day 50-55 both sexes attained adult enzyme levels. STII was the major enzyme in all immature animals. STIII was also present, but STI was absent. In male rats STIII activity began to rise by day 30. Soon after, STII activity began to drop. By day 55 adult male patterns developed. STI was the major enzyme in females by day 30. In ensuing days all 3 enzyme levels rose, until by day 50 adult enzyme patterns and levels were attained. The data suggest that the ovaries stimulated production of all 3 sulfotransferases and that the testes suppressed production of STII (and perhaps STI). Preliminary studies with gonadectomized rats supported the suppressive role of the testes, but suggested that the ovaries were not the only factor controlling sulfotransferase production in female rats.     

Iodothyronine deiodination in rats with severe non-thyroidal illness

To investigate the iodothyronine metabolism in non-thyroidal illness (NTI), thyroidectomized male Wistar rats bearing the hypercalcemic Walker sarcoma 256 were substituted with, respectively, 2.3 and 11.5 mumol T4/100 g body weight by daily ip injection. Serum T4 and T3 concentrations of euthyroid and hyperthyroid tumor-bearing animals markedly decreased to a nadir at day 8 after tumor implantation: serum T4 fell to, respectively, 43% (euthyroid) and 26% (hyperthyroid) of initial values, serum T3 to 19% (euthyroid) and 26% (hyperthyroid). A measurable serum rT3 concentration could not be detected before and after tumor implantation. In vitro deiodination of T4 to T3 in liver homogenates of the sacrificed animals was not significantly reduced in Walker rats compared with control animals. The activity of T4 deiodinase was significantly induced in hyperthyroid controls (180%) as well as in hyperthyroid Walker rats (155%) in spite of low serum concentrations of T4 and T3. This enzyme induction was even more pronounced in animals whose treatment with high T4 doses was started after tumor implantation. In these rats the serum concentrations of free fatty acids were increased to about 200% of controls. Our data suggest that 1. the fluctuations of iodothyronine serum concentrations in NTI are mainly independent of thyroidal secretion, and 2. the intracellular iodothyronine levels in livers of severely sick animals with different thyroid function are not greatly altered by NTI, in spite of markedly decreased total serum levels.     

Hypometabolic effect of 3,3',5'-triiodothyronine in chickens: interaction with hypermetabolic effect of 3,5,3'-triiodothyronine

The effect of 3,3',5'-triiodothyronine (rT3) and 3,5,3'-triiodothyronine (T3) on O2 consumption in 1-day-old chickens was studied. The birds were divided into five groups, each of six chickens: (1) control--without injection; (2) control--injected with 100 microliters of solvent (0.01 N NaOH in saline); (3) injected with 10 micrograms rT3/chicken; (4) injected with 0.5 micrograms T3/chicken; and (5) injected with 10 micrograms rT3 + 0.5 microgram T3/chicken. O2 consumption was measured using a Kipp & Zonen diaferometer at neutral temperature (30 degrees) 0, 1, 2, 3, and 4 hr after injection of hormones. Corresponding groups of other chickens served only for blood collection. rT3 and T3 were measured by radioimmunoassay. Reverse T3 decreased O2 consumption by 10.87%. Contrary to this, T3 increased O2 consumption by 29.41%. Reverse T3, injected together with T3, interacted with the hypermetabolic effect of T3 up to 2 hr after injection; then, O2 consumption started to increase, and was about 16.7% higher compared with the basal level 3 hr after injection. The blood plasma level of rT3 increased about 29-fold at the first hour after injection, without changes in the basal level of T3. Administration of T3 increased its level 6-fold 2 hr after injection, which was accompanied by a gradual decrease in the basal level of rT3 (3.7-fold) 4 hr after injection. Administration of rT3 + T3 increased the rT3 level 30-fold at 2 hr and the T3 level 1.7-fold at the first hour after injection. Thus, rT3 acts hypometabolically and interacts with the hypermetabolic effect of T3; administration of T3 lowered the basal level of rT3; and the plasma level of T3 did not change after administration of rT3.