Thyroid hormone metabolism in primary cultured rat hepatocytes. Effects of glucose, glucagon, and insulin.

K Sato, J Robbins

J Clin Invest. 1981;68(2):475-483. https://doi.org/10.1172/JCI110278.

Research Article

Primary cultured adult rat hepatocytes were used to study regulation of thyroid hormone deiodination. Control studies showed that these cells survived for at leas 4 d, during which time they actively deiodinated both the phenolic (5'-) and non-phenolic (5-) rings of L-thyroxine (T4),3,5,3'-triiodo-L-thyronine, and 3,3',5'-triiodothyronine. Increasing the substate concentration caused a decrease in fractional iodide release and a corresponding increase in conjugation with sulfate and glucuronide. Propylthiouracil strongly inhibited the 5'-deiodinase activity and caused only a slight decrease in 5- deiodinase activity. Thus, these monolayer-cultured cells preserved many of the properties of normal hepatocytes. Incubation with combinations of insulin, glucagon, and/or glucose for 5 h showed that insulin stimulated T4 5'- deiodination, whereas glucagon inhibited the insulin stimulation but had no effect in the absence of insulin. Glucose had no effect and did not alter the effect of the hormones. The insulin-enhanced deiodination increased between 1 and 5 h, which suggests that the previous inability to demonstrate an insulin effect was due to the short survival of the in vitro liver systems used in those studies. The present data suggest that the inhibition of T4 5'-deiodination observed during fasting, and its restoration by refeeding, may be related to the effects of feeding on insulin and glucagon release rather than on glucose per se.

Find the latest version: https://jci.me/110278/pdf Thyroid Hormone Metabolism in Primary Cultured Rat Hepatocytes

EFFECTS OF GLUCOSE, GLUCAGON, AND INSULIN

KANJI SATO and JACOB ROBBINS, Clinical Endocrinology Branch, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205

A B S T R A C T Primary cultured adult rat hepatocytes the conversion of L-thyroxine (T4)' to biologically active were used to study the regulation of thyroid hormone 3,5,3'-triiodo-L-thyronine (T3) is decreased in fasting deiodination. Control studies showed that these cells and increased by refeeding, especially of carbohydrate survived for at least 4 d, during which time they ac- (1-5). This regulatory system may operate in starvation tively deiodinated both the phenolic (5'-) and non- to conserve limited energy sources for essential bio- phenolic (5-) rings of L-thyroxine (T4),3,5,3'-triiodo- chemical processes. Although, in the starved rat, thy- L-thyronine, and 3,3',5'-triiodothyronine. Increasing roid gland secretion is also diminished (6, 7), a number the substrate concentration caused a decrease in frac- of studies have demonstrated decreased production of tional iodide release and a corresponding increase in T3 from T4 in the liver. Various hypotheses have been conjugation with sulfate and glucuronide. Propyl- proposed to explain this altered production of T3; thiouracil strongly inhibited the 5'-deiodinase activity namely, decreased cofactor activity in the cytosol and caused only a slight decrease in 5-deiodinase ac- (8-11), such as nonprotein sulfhydryl compounds tivity. Thus, these monolayer-cultured cells preserved (mainly glutathione), decreased NADPH concentra- many of the properties of normal hepatocytes. Incuba- tion and decreased glutathione transhydrogenase (12), tion with combinations of insulin, glucagon, and/or a decreased amount of deiodinases (13), or decreased glucose for 5 h showed that insulin stimulated T4 5'- cellular uptake of T4 (14). Most of these studies, how- deiodination, whereas glucagon inhibited the insulin ever, were performed using crude liver homogenate stimulation but had no effect in the absence of insulin. under quite unphysiological conditions. Although a Glucose had no effect and did not alter the effect of few studies used liver slices (15) and perfused liver the hormones. The insulin-enhanced deiodination in- (14), their limited viability, several hours at most, make creased between 1 and 5 h, which suggests that the pre- them unsuitable in an investigation of the regulatory vious inability to demonstrate an insulin effect was due mechanism of the starvation effect, which takes place to the short survival of the in vitro liver systems used in days rather than hours (1-5). This disadvantage can in those studies. The present data suggest that the in- be resolved by using primary cultured adult rat hepato- hibition of T4 5'-deiodination observed during fasting, cytes (16), which also offer the advantage of cellular and its restoration by refeeding, mnay be related to the homogeneity, the opportunity to conduct experiments effects of feeding on insulin and glucagon release rather with homologous cells from a single rat, and especially than on glucose per se. the ability to study the effect of various nutrients or hormones without the secondary hormonal responses INTRODUCTION from other organs, as can occur in whole animial studies.

The mammalian organism has an apparent adaptation I Abbreviationis used itn this paper: Arg(-)DV imiediumii, ar- mechanism for thyroid hormone metabolism by which ginine-free Dulbecco-Vogt medium; comiiplete Arg(-)DV medium, Arg(--)medium supplemented with bovine in1sulin Dr. Sato is on leave from the Third Department of Initernial and hvdrocortisone hemisuceinate; EE medium, Eagle's miini- Medicine, Uniiversity of Tokyo. His presenit address is The mal essential medium with Earle's balaneedl salt solution; Second Department of Medicine, Tokyo Womien 's MIedical complete EE mediumii, EE mediumll slippleimieintedl with in- College, Ichigaya-Kawadacho, Shinjuku, Tokyo, Japain. sulin and hydrocortisone hemnisuccinate; PTU, 6-ni-propyl-2- Received for publication 30 December 1980 and in re- thiouracil; rT3, 3,3',5'-triiodo-L-thyronine; Ti, 3,5,3'-triiodo- vised forml 13 April 1981. L-thyroniine; T4, L-thyroxine.

The Journal of Clinical Itvestigation Volume 68 August 1981 475-483 475 Previously, we reported (17) that primary cultured hepatocytes from a single rat (200-300 g) were suspended in adult rat hepatocytes maintained 5'- and 5-deiodinating complete Arg(-)DV medium (cell density, 0.5-0.6 x 106/ml). Aliquots (5-ml) of the cell suspension were added to 25 cm2 activities for at least 4 d. In contrast to poorly differen- Falcon plastic flasks (Falcon Labware, Div. of Becton, Dickin- tiated monkey hepatocarcinoma cells (NCLP-6E), son & Co., Oxnard, Calif.), and kept at 37°C under 5% CO2 these well-differentiated rat hepatocytes preserved en- and 95% air; at this optimal cell density the recovery efficiency zymes of the urea cycle (conversion of [3H]ornithine to (ratio of attached cells to total cells 24 h after plating) was [3H]arginine), 20-30%. In the presence of supraphysiological concentrations the transsulfuration pathway (gluta- of insulin and hydrocortisone, and without changing the thione synthesis from methionine), and sulfo- and medium, the number of cells increased after 3 d to -1.5 glucurono-conjugation (17), and thus are considered to x 106/flask. The hepatocytes became flattened, polyhedral in be a more suitable model for studying thyroid hormone shape, and formed trabeculae which reached confluence in metabolism. By culturing the hepatocytes in cys- some places. After 4 d, the cells began to degenerate, be- coming round and granular, and losing adherence to the plas- tine and methionine-deficient medium, thereby de- tic. By day 7 almost all cells became detached. creasing the intracellular glutathione concentration to When hepatocytes were grown in complete EE medium, <10% of the control (17), we also demonstrated there was better recovery efficiency (>30%) at 24 h, and a that thyroid hormone deiodination is not modulated by greater number of cells survived after 3 d (-2 x 106/flask); nonprotein levels in but hepatocytes grown in complete EE medium started to de- sulfhydryl the liver. generate at the beginning of the 4th day after plating, and Using these well-differentiated adult rat hepatocytes, nonparenchymal or nonarginine-synthesizing cells could also we investigated the effects of glucose, glucagon, and in- grow. Therefore, arginine-free, ornithine-supplemented Dul- sulin. We found that thyroid hormone metabolism was becco-Vogt medium was usually used. not affected by glucose alone, but rather by insulin, Metabolism of various concentrations of T4, T3, and 3,3',5'- and triiodithyronine (rT3) added to the medium. Cells grown that the stimulatory effect of insulin was abolished in complete EE medium for 2 d were washed three times with by glucagon. Furthermore, the insulin effect was evi- 5 ml of Dulbecco buffer, and 3 ml of serum-free EE medium dent at 10 ng/ml, a concentration found in the portal was added. After a 1-h preincubation at 37°C under 5% C02 vein (18). This suggests that the fasting- and feeding- and 95% air, T4, T3, or rT3 dissolved in 30 ,ul of 5 mM NaOH induced alteration of thyroid hormone metabolism may was added to a final concentration of 1 nM-10 ,AM, followed by the addition of -300,000 cpm of [3',5'-125I]T4, [3'1_25I]_ be regulated by these hormones. T3, or [3',5'-_25I]rT3, respectively. In some experiments, [3,5-'25I]T4 or [3,5-1251]T3 was used. After an additional 3 and 15 h incubation, 0.5 ml of medium was taken and 10 ,ug of METHODS cold T4, T3, or rT3 dissolved in 10 ,ul of alkalinized n-butanol was added as carrier. After lyophilization of the samples, [3',5'-125I]T4 (--1 mCi/lug) and [3'-1251]T3 (-1 mCi/4g) were 125I-labeled iodothyronine metabolites were analyzed by thin- purchased from New England Nuclear, Boston, Mass. [3,5- layer chromatography (TLC) with the solvent system of n- 125I]T4 (-2 mCi/,Lg) and [3,5-1251]T3 were synthesized accord- butanol saturated with 2 N NH4OH, as described before (22). ing to Cahnmann's methods (19, 20). All radioactive tracers Effect of PTU on T4 and T3 metabolites in the medium. stored >10 d (in ethanol at 4°C) were purified by ion-exchange 2 d after growing the hepatocytes in complete Arg(-)DV column chromatography (0.9 x 10 cm, Dowex 50W-X4, 30-35 medium, spent medium was aspirated, and the cell monolayer ,um, Bio-Rad Laboratories, Richmond, Calif.), followed by was washed three times with 5 ml of Dulbecco-Vogt buffer passing through a short column (0.9 x 1.0 cm) of Dowex 50W- (pH 7.4). Then, 3 ml of serum-free Arg(-)DV medium contain- X4 to remove 125I- as described previously (17). Other ma- ing various concentrations of PTU (0.001-0.1 mM) was added. terials were 6-n-propyl-2-thiouracil (PTU) (Sigma Chemical After a 1-h preincubation at 37°C under 5% CO2 and 95% Co., St. Louis, Mo.), bovine insulin (Sigma), bovine glucagon air, [3',5'-125I]T4 [3,5-1251]T4, [3'-125I]T3 or [3,5-125I]T3 dis- (Calbiochem-Behring Corp., American Hoechst Corp., San solved in 50 ,ul of saline was added to a final concentration of Diego, Calif.), and hydrocortisone hemisuccinate (Sigma). -0.1 nM. After an additional 3-h incubation, 0.5 ml of medium Primary culture ofadult rat hepatocytes. Culture medium was taken and [125I]iodothyronine metabolites released into routinely used was arginine-free Dulbecco-Vogt medium the medium were analyzed by TLC as above. containing 10 mM Hepes, 15% dialyzed fetal bovine serum, T4 metabolites in the cells. In some experiments, intracel- streptomycin sulfate (100 ,ug/ml), penicillin (100 U/ml), and or- lular T4 metabolites at 3 h incubation were analyzed after nithine (0.4 mM) [Arg(-)DV medium], which was supple- [125I]T4 metabolites were extracted with n-butanol by a modi- mented with bovine insulin (10 ,ug/ml) and hydrocortisone fication of Flock's method (23). After an aliquot of medium hemisuccinate (10 ,tg/ml) [complete Arg(-)DV medium]. In was taken for analysis, the rest of the medium was aspirated. this complete Arg(-)DV medium, only cells containing en- Cell monolayers were washed with 5 ml of ice-cold Dulbecco zymes for the urea cycle (hepatocytes) can grow (21). buffer (pH 7.4). Then 1 ml of 0.1% sodium dodecyl sulfate In some cases, Eagle's minimal essential medium with solution containing 1 mM PTU, 5 mM diamide, 1 mM N- Earle's balanced salt solution containing 10% undialyzed fetal ethylmaleimide, and 10 ,M T4 was added and incubated at bovine serum, 10 mM Hepes, 2 mM glutamine, 1% non- 37°C for 1 h. Cell lysates were acidified with 0.1 ml 4 N HCl, essential amino acids, streptomycin sulfate (100 ,ug/ml), and and the acidified samples were extracted twice with 2 ml ofn- penicillin (100 U/ml) (EE medium) was used and was sup- butanol. Then 4 ml of chloroform was added to the combined plemented with insulin (10 ,ug/ml) and hydrocortisone hemi- n-butanol extract and mixed well. The turbid mixture was ex- succinate (10 ,ug/ml) (complete EE medium). tracted with 2 and 1 ml of 2 N NH40H, successively. The Primary culture of adult rat hepatocytes was performed combined extracts were lyophilized and T4 metabolites were by the method of Leffert et al. (16X, with slight modifications analyzed by TLC with 5 ,ug of T4, T3, and rT3 as carriers and as described previously (17). In brief, collagenase-dispersed markers. The recovery of 1251 was -80%. When 1 ml of [125 ]T4-

476 K. Sato and J. Robbins containing medium was treated in the same way, -1% of 1251 comigrated with T3, 1% with rT3, and 94% with [125I]T4. [3' 5_1251]T4 Effect of glucose, glucagon, and insulin. Hepatocytes were grown for 2 d in complete Arg(-)DV medium. Mono- layer cells were washed three times with 5 ml of Dulbecco buffer, then incubated for 15 h with 3 ml of serum-free Arg(-)DV medium. (In this serum-free, hormone-free Arg(-)- DV medium, hepatocytes usually survived for up to 48 h, but sometimes started to degenerate after 36 h. All the experi- ments were therefore performed within 3 d after plating.) Then 30 Al of insulin or glucagon dissolved in 10 mM HC1 was added to a final concentration of 10 ng/ml to 10 ,ug/ml. [3,1251J T3 For control cells, 30 ,ul of 10 mM HC1 was added. After a 5-h zw preincubation at 37°C under 5% CO2 and 95% air, [3',5'-125I]_ D 14 T4 dissolved in 30 Ml of 0.9% NaCl was added, and after an U, additional 3-h incubation, [125I]T4 metabolites in the lyoph- 0 U ilized medium were analyzed by TLC as above. H To investigate the fasting and feeding effects on thyroid z hormone metabolism, the effects of glucose, glucagon, and in- sulin in various combinations were studied after the hepato- cytes were grown for 2 d in complete Arg(-)DV medium. w Washed monolayer cells were then incubated with 3 ml of 0U 1I serum-free, glucose-free Arg(-)DV medium for 15 h. Insulin, and/or glucagon were added to a final concentration of 5 [3',5 1251]rT3 Ag/ml for each. For control cells, 30 Al of 10 mM HCI was added. In some flasks, 30 ,ul of glucose was added to a final concentration of 250 mg/dl. After a 5-h incubation, [3',5'- '251]T4 dissolved in 30 A1 of saline was added to a final con- centration of -0.1 nM, and, after an additional 3-h incubation, [1251]T4 metabolites secreted in the medium were analyzed by TLC. In this serum-free, glucose-free medium, cells ap- peared intact for >24 h, but some degeneration started by 40 h. 24 Therefore, all the experiments were completed within h e,jO.lnM 10nM 1wM rO.inM iOnM 1vM after changing the medium, or within 72 h after plating. After the medium was taken for analysis of [125I]T4 metabo- SUBSTRATE CONCENTRATION lites, washed monolayer cells were treated with 1 ml of FIGURE 1 Thyroid hormone metabolism at various substrate 0.05% trypsin dissolved in Ca", Mg++-free Dulbecco buffer concentrations. Hepatocytes were grown for 2 d in complete containing 0.5 mM EDTA. After 2-3 min incubation at room EE medium. Monolayer cells were incubated with 3 ml of temperature, 3 ml of EE medium was added and the number serum-free EE medium. After a 1-h preincubation at 37°C, of cells was counted in a hemocytometer. Since there was no T4, T3, or rT3 dissolved in 30 Al of 5 mM NaOH was added to a significant difference in cell numbers in control, glucagon, final concentration of 1 nM-10 ,M followed by the addition and insulin (-1.5 x 106 flask), the results were expressed as of -300,000 cpm of [3',5'-125I]T4, [3'-125I]T3, or [3',5'- percentage of total 1251. 125I]rT3. After an additional 3- and 15-h incubation, medium To investigate a more prolonged period of T4 metabolism was taken for analysis. Note that major metabolites are 125I- approaching the steady-stage condition, [125I ]T4 metabolism (@) at the lower substrate concentration, but are the conjugates was investigated in the presence of serum in a few experi- (dotted and broken lines) at the higher concentrations. Per- ments. After the cells were grown in complete Arg(-)DV centage of total 1251 is indicated as T4 (U), T3 (A), rT3 (V), C, (glu- medium for 24 h, medium was replaced with 3 ml of Arg(-)- curonoconjugates, A), and C2 (sulfoconjugates, 0). DV medium containing 10% dialyzed serum, streptomycin (100 ,ug/ml), penicillin (100 U/ml), 10 mM Hepes, various con- centrations of glucose (0, 1, and 8 mg/ml), and -500,000 cpm of [3',5'-_25I]T4. The T4 concentration in the dialyzed fetal tion 1-10 ,uM. The same metabolites were obtained bovine serum was 9,ug/dl. As a cell-free control, [1251]T4 was when [3,5-125I]T4 was used as a tracer (data not shown). incubated in the same medium in a cell-free flask. After a 48-h incubation, 1 ml of medium was taken, and ['251]T4 metabo- The ratio of 1251- released from [3,5-1251]T4 to that from lites were extracted according to Flock's (23) method, and [3'5 _125I]T4 was -1 at all substrate concentrations. analyzed by TLC. The recovery of radioiodine was >80%. Since little iodide was released from either ring of T4 Nonspecific deiodination of [125I]T4 was 1.3%, with < 1% of T3 at the higher concentrations, the bands designated as and rT3 contaminants. C1 (Rf 0.04) and C2 (Rf 0.14) were nondeiodinated T4 derivatives. The treatment of isolated C1 and C2 RESULTS bands with 3-glucocuronidase or sulfatase revealed Effect of T4, T3, and rT3 concentration on thyroid that these were composed mainly of T4 glucuronide and hormone metabolism (Fig. 1). The major metabolite T4 sulfate, respectively. of [3',5'-125I]T4 at low concentrations, as reported Similarly, the major metabolites of [3'-1251]T3 were previously (17), was 1251-, whereas T4-conjugates were iodide at low concentrations, but T3 conjugates at the found with little iodide release at higher T4 concentra- higher concentrations. When [3,5-125I]T3 was used, a

Thyroid Hormone Metabolism 477 qualitatively similar chromatogram pattern was ob- contrast to monkey hepatocarcinoma cells (17), can tained with 4% 125I- release at 10 ,uM, which suggests synthesize sulfate endogenously. that the Cl and C2 bands were T, conjugates. The ratio PTU effect on T4 and T3 metabolism. As shown in of 125I- released from [3,5-125I]T3 to that from [3'-1251]T3 Table I and Fig. 2, PTU inhibited 5'-deiodination of at l5h was 1.9, 3.3,and 3.1, at 0.1, 1, and 10 ,uM, [125I]T4 in a dose-dependent fashion. On the other hand, respectively, which suggests that the major deiodination 5-deiodination of [125I]T4 was only slightly affected pathway of T3 was to 3,3'-diiodo-L-thyronine (3,3'-T2), even at 0.1 mM PTU. The marked inhibitory effect on 5'- which was identified in the autoradiogram exposed >3 deiodination gave rise to an increased amount of rT3. As d (data not shown). The treatment of C1 and C2 bands expected, slightly more rT3 was observed in metabolism with 3-glucuronidase or sulfatase revealed that these of [3',5'-125I]T4 than with that of [3,5-'25I]T4. The bands were mainly T3 glucuronide, and T3 sulfate (with inhibition of deiodination ofT4 was accompanied by an small amounts of 3,3'-T2-sulfate), respectively. In con- increase in T4 conjugates. This was even more striking trast to the T4 conjugates, much more T3 sulfate was after further incubation for 5 h (Fig. 2A). formed, compared with the glucuronoconjugate. Analogous with its effect on [2l5I]T4 metabolism, PTU [3',5'-125I]rT3 metabolism also proceeded rapidly also markedly inhibited outer ring deiodination of through outer ring deiodination at the lower concentra- [125I]T3 (Fig. 2B). Little inhibitory effect was observed tions, but, at the higher concentrations, it was sulfo- and on inner ring deiodination of T3, but when the glucurono-conjugated. percentage of 125J released from [3,5-125I]T3 was It can be seen that thyroid hormone 5'-deiodination measured, a slight but significant inhibitory effect was proceeds rapidly in the order of rT3 > T3 > T4 (Fig. 1 at observed as it was with T4. The striking inhibition of 3 h), and that sulfoconjugation takes place in the order phenolic ring deiodination and the slight inhibition of ofT3> rT3> T4, with reciprocally decreased formation nonphenolic ring deiodination of T3 gave rise to of glucuronoconjugates (T3 < rT3 < T4) (Fig. 1 at 15 h). increased formation of conjugates, especially of Using T3, the best substrate for phenol sulfotransferase, sulfoconjugates (Table I and Fig. 2). [125I]T3 metabolism was studied in the absence of T4 metabolites in the cells. Intracellular T4 metabo- exogenous sulfate (streptomycin sulfate) in the medium; lites were analyzed after incubation for 3 h with -0.1 no change in T3 sulfate formation was detected. This nM [125I]T4. A slight but distinct amount of T3 (2-3%) indicates that primary cultured rat hepatocytes, in was detected. Radioactivity comigrated mostly with T4

TABLE I PTU Effect on [125I] T4 Metabolism ['2lI]T4 metabolites PITU T4 rT3 C2 Cl Origin

mM % of total [3',5' 125I]T4 0 (n = 4) 32.2+2.4 56.4± 1.9 3.7+0.8 5.8± 1.4 3.0±0.9 0.4+0.2 0.001 (n = 3) 9.3±2.7* 57.8+8.2 11.5+1.2t 13.9+3.2 7.0±+1.8 0.5+0.1 0.01 (n = 4) 4.9±0.74 60.9+8.8 14.4±2.6* 12.9+4.3 6.4+2.6 0.3+0.1 0.1 (n = 4) 3.5±0.44 62.6+8.5 13.8±2.5* 12.3+±3.6 7.2-+2.8 0.2+±0.1 [3,5 125I]T4 0 (n = 3) 27.7±+1.3 58.2+2.4 2.0+0.7 8.6±+1.7 2.8±0.4 0.7±0.1 0.001 (n = 2) 20.2 59.1 8.9 8.3 3.1 0.4 0.01 (n = 3) 19.5±1.0t 61.0±0.3 8.2±0.3* 6.9±0.5 3.2±0.9 0.9±0.3 0.1 (n = 3) 16.6±2.2* 65.3±1.9* 7.7±0.1* 6.0±0.2 3.7±0.9 0.7±0.2 Rat hepatocytes were grown in complete Arg(-)DV medium for 2 d. Then medium was replaced with 3 ml of serum-free Arg(-)DV medium, and PTU was added. After a 1-h pre- incubation, [3',5'-125I]T4 or [3,5-1251]T4 was added to a final concentration of 0.1-0.2 mM. After an additional 3-h incubation, 0.5 ml of the medium was taken for analysis of [125I]T4 metabolites released from the cells. No T, band was detected on the autoradiographs, and the T, area was not counted in these experiments. Cl and C2 represent glucurono- and sulfo- conjugates, respectively. Data are mean±SEM. Statistical analyses were performed by paired t test. * P < 0.05. 4 P < 0.01.

478 K. Sato and J. Robbins is primarily ascribed to an increased rate of deiodina- tion; conjugation was little affected. Glucagon alone had no effect on T4 metabolism. In fact, in four of seven experiments, glucagon appeared to show a slight inhibitory effect on 5'-deiodination, but this was not statistically significant (Table II). There was also no ef- fect on the amount of T3 or rT3 produced. *0 4U-jT3 There was no difference in [3',5'-1251]T4 metabolism '-SULFATES between glucose-fed and glucose-deprived groups, _ O * _ w _ _ which suggests that fasting-induced, and feeding-re- -GLUCURONIDES versed alterations of thyroid hormone metabolism are -ORIGIN modulated by glucoregulatory hormones but not by carbohydrate alone.

B 0- To investigate a prolonged effect of glucose-depri- ow 4 'm 40 4W vation, cells were grown in aglycemic, normoglycemic (1 mg/ml), and hyperglycemic (8 mg/ml) medium for 48 h in the presence of 10% dialyzed serum containing [3',5'-1251]T4. The cell number at 3 d was -1.5 x 106/ flask. Major [1251]T4 metabolites in the normoglycemic _t - _ 4_ _db -SULFATES medium were 1251- = 22.8%, rT3 = 4.9%, Cl = 5.3%, -GLUCURONIDES and C2 = 3.5%, with little T3 formation (<1%). Hyper- I 0 O o0 Oo ORIGIN glycemic medium caused no change. A slight decrease PTU 0 0.001 0.01 0.1 mM in 125I- release was observed in aglycemic medium (19.3%), but because the number of cells grown in glu- FIGURE 2 Effect of PTU on thyroid hormone metabolism. cose-free medium was -10% less than that in the glu- Hepatocytes were grown for 2 d in complete Arg(-)DV cose-fed the medium. Then monolayer cells were incubated with 3 ml of cells, interpretation of this change is serum free Arg(-)DV medium containing various concentra- difficult. tions of PTU (0.001-0.1 mM). After a 1-h preincubation at 37°C, [3,5-125I]T4 (inner-ring labeled, I) or [3',5'-1251]T4 (outer DISCUSSION ring-labeled, 0) (Fig. 2A) or [3,5-125J]T3 (I) or [3'-1251]T3 (0) (Fig. 2B) were added to a final concentration of -0.1 nM. Most of our experiments were carried out at a concen- After an additional 5-h incubation for T4 and 3 h for T3, 0.5 tration of -0.1 nM T4 in the medium, since this is close ml of medium was taken and metabolites were analyzed by TLC. Note that PTU markedly inhibits outer ring deiodination to the free T4 concentration in rat serum (24). In our of T4 and T3. system, -50% of T4 was metabolized in 3 h by 1.5 x 106 cells in 3 ml of serum-free medium containing 0.1-0.2 nM T4 (0.1-0.2 ng of T4/1.5 x 106 cells per 3 h (-70%), accompanied by 1- (-4%), rT3 (-10%), Cl- (-5%), and C2(-4%). 22 Effect of glucose, glucagon, and insulin. As shown - 20 in Fig. 3, preincubation with insulin (10 ng/ml-10 ,ug/ co ml) for 5 h caused marked stimulation of 5'-deiodina- a 18 y tion of [3',5'-1251]T4, whereas glucagon had no effect &H 16 at any concentration. This stimulatory effect of insulin 14 was less marked when [125I]T4 was added after only a 1-h incubation with insulin. 0 101 102 103 104 Next, the effects of glucose, glucagon, and insulin Insulin or Glucagon (ng/mI) were studied in hepatocytes incubated in serum-free, glucose-free Arg(-)DV medium for 15 h. 5 h after the FIGURE 3 Effect of insulin and glucagon on 5'-deiodination of [3',5'-125 ]T4. Hepatocytes were grown for 2 d in com- addition of glucose (2.5 mg/ml), glucagon (5 ,ug/ml), plete Arg(-)DV medium. Monolayer cells were washed and/or insulin (5 ,ug/ml), [3',5'-125I]T4 metabolites and incubated with 3 ml of serum-free, hormone-free secreted in the medium during 3 h were analyzed by Arg(-)DV medium for 15 h. Then 30 IAl of insulin (-) or gluca- TLC. As shown in Table II, insulin significantly in- gon (A) dissolved in 10 mM HCI was added to a final concentra- creased 5'-deiodination, and this stimulatory effect was tion of 10 ng/ml-10 ,ug/ml. After a 5-h preincubation, [3',5'- 125I]T4 was added to a final concentration of -0.1 nM, and after inhibited by glucagon. This occurred either in the pres- an additional 3-h incubation, [1251]T4 metabolites in the ence or absence of glucose. It should be pointed out medium were analyzed by TLC. Data are means of duplicate that the stimulatory effect of insulin on T4 metabolism determinations.

Thyroid Hormone Metabolism 479 TABLE II Effects of Glucose, Glucagon, and Insulin on [3',5'-1251]T4 Metabolism ["'5I]T4 metabolites

Glucose Glucagon Insulin I T3 T4 rT, C2 C, Origin % of total (-) (-) (-) 25.5 0.5 54.0 4.8 9.4 5.2 0.3 ±2.4 ±0.1 ±3.6 ±0.8 ±1.3 ±1.3 ±0.1 (-) (+) (-) 24.1 0.5 55.1 4.9 9.4 5.7 0.3 ±2.8 ±0.1 ±5.3 ±0.6 ±1.6 + 1.6 ±0.1 (-) (-) (+) 32.8* 0.6 44.4t 5.3 10.8 5.7 0.3 ±1.7 ±0.1 ±2.8 ±0.4 ±1.4 ±1.5 ±0.1 (-) (+) (±) 26.5§ 0.5 52.15 5.1 9.8 5.5 0.4 ±2.6 ±0.1 ±4.7 ±0.6 ±1.6 ±1.4 ±0.1 (+) (-) (-) 26.7 0.5 52.1 4.7 9.7 5.7 0.3 ±1.8 ±0.04 ±3.7 ±0.5 ±1.3 ±1.7 ±0.1 (+) (+) (-) 24.0 0.5 56.0 4.2 9.3 5.4 0.3 ±2.5 ±0.04 ±5.2 ±0.4 ±1.6 ±1.7 ±0.2 (+) (-) (+) 32.6* 0.5 44.8t 5.3 10.8 5.7 0.4 +2.1 ±0.1 ±3.1 ±0.4 ±1.3 ±1.6 ±0.1 (+) (+) (±) 26.55 0.4 52.15 5.1 9.8 5.8 0.3 ±2.6 ±0.1 ±4.9 ±1.0 ±1.6 ±1.8 ±0.1 Cells were grown for 2 d in complete Arg(-)DV medium. The medium was replaced with hormone-free, glucose-free, protein-free Arg(-)DV medium and incubated for 15 h. Then, glucose, glucagon, and/or insulin were added to 2.5 mg/ml, 5 ,tg/ml, and 5 ,ug/ml, respectively. After a 5-h incubation, [3',5'-'25IJT4 was added to a final concentration of-0. 1 n M. After an additional 3-h incubation, 0.5 ml of medium was taken, and [125I]T4 metabolites were analyzed by TLC. Data are means±SEM of seven experiments. C, and C2 represent glucurono- and sulfoconjugates, respectively. Statistical analysis by paired t test are as indicated in footnotes. * P < 0.001 when compared with the control. t P < 0.01 when compared with the control. § P < 0.05 when glucagon(+)-insulin(+) groups are compared with glucagon(-) insulin(+) groups. or 0.06-0.12 Lg/108 cells per 24 h). This can be com- phenomenon was recently elucidated by Sekura pared with the rate of T4 metabolism in the liver in vivo. et al. (28, 29), who purified sulfotransferase from It is generally accepted that 2 ,tg of T4/100 g of body rat liver (28) and showed that the enzymes had an af- weight is required to maintain a rat in a euthyroid state finity for T3, rT3, and T4, in this order (29). The poor (25). On the assumption that 30% of T4 is metabolized activity of T4 as a substrate for the phenolsulfotrans- in the liver, which makes up -4% of total body weight, ferase probably causes it to take the alternate conjugat- and that 1 g of liver contains 1.3 x 108 hepatocytes (26), ing pathway. Nothing, however, is known about the re- hepatocytes in vivo metabolize -0.12 ,mg of T4/108 cells activity of glucuronotransferase with T4 analogues. In per 24 h. Therefore, the present experimental condition contrast to monkey hepatocarcinoma cells (17, 22), the is a suitable model to study the regulatory mechanism hepatocytes were able to synthesize T3 sulfates in the of thyroid hormone metabolism. At the lower concen- absence of exogenous sulfate, which indicates that the tration of T4, the major metabolite was 125P-; increasing rat hepatocytes synthesize sulfate endogenously by the the T4 concentration to a supraphysiological level (1-10 cysteine-oxidizing pathway (30). The greater percent- ,uM), shifted the metabolism from the deiodinating age of T3 sulfate found at 3 h compared with 15 h at pathway to the detoxification pathway involving sulfo- 0.1-10 nM (Fig. 1) suggests that the sulfoconjugate and glucurono-conjugation, as reported in vivo (27). is slowly hydrolyzed by an intracellular sulfatase (31). It is of interest to note the relative amounts of sulfo- As is well known from both in vivo and in vitro data and glucurono-conjugation for the three iodothyronines (32, 33), PTU efficiently inhibits outer ring deiodina- studied. For sulfation, these were T3 > rT3 > T4. This tion of T4 and T3. PTU also has a slight inhibitory ef-

480 K. Sato and J. Robbins fect on inner ring deiodination of T4 and T3 (34, 35), nings et al. (14) using liver slices and perfused liver, which results in decreased serum T3 concentration with respectively, to obtain a positive insulin effect might be reciprocally increased rT3 concentration (36). These in- ascribed to the short incubation time inherent in their hibitory effects on T4 metabolism increase T4 metabo- methodologies. lism through the conjugating pathways (37). These phe- Generally, insulin and glucagon are considered to nomena can be visualized in Fig. 2. It should be pointed have antagonistic effects on hepatic metabolism. One out that the PTU concentration added in the medium of the few exceptions, however, is the regulation of was within the range achieved in human subjects tak- liver cell growth; glucagon and insulin synergistically ing 200 mg PTU per os (0.5-10 ,ug/ml or 0.003-0.06 stimulate DNA synthesis and cell proliferation of neo- mM) (38). natal or adult rat hepatocytes in primary culture (46,47) The most interesting findings in the present experi- and in vivo (48). Glucagon showed no effect on cell ments are the effects of glucose, glucagon, and insulin growth in our culture system, and our finding of its an- on thyroid hormone metabolism. Our data clearly show tagonistic effect suggests that insulin and glucagon are that insulin increased 5'-deiodination of T4 after several probably not acting through an effect on cell growth. hours of incubation. The insulin effect was more evi- Insulin is a very potent cell growth factor for hepato- dent after a 5-h incubation than after a 1-h incubation, cytes, however, and this possibility should therefore be and this effect was antagonized by glucagon. The insu- taken into consideration, even though the number of lin effect was observed at 10 ng/ml, a physiological cells did not change significantly between control and concentration of insulin in the portal vein (18), which insulin-treated cells within the time-course of the suggests that the hormone might regulate thyroid hor- present experiments (8-h maximum). mone metabolism at least in the liver in vivo. Since fast- As reported previously (17), 5'- and 5-deiodination ing is accompanied by hypoinsulinemia and hyper- proceeded always to the same extent in these primary glucagonemia, and since refeeding, especially of carbo- cultured rat hepatocytes. Therefore, T3 production hydrate, is accompanied by hyperinsulinemia and should have occurred, and T3 was in fact demonstrated hypoglucagonemia (18), the present in vitro data sup- intracellularly. A curious finding in the present series port the hypothesis that thyroid hormone metabolism is of experiments, however, was that we were able to modulated by hormones rather than by glucose alone. detect rT3 distinctly but could hardly detect T3 in the Recently, Balsam and Ingbar (15) reported that insulin culture medium (Tables I, II). We postulated previ- administration to streptozotocin-induced diabetic ously (17) that a nonequilibrium condition between T4 rats normalized the diminished T3 generation from T4 stored in the cell and [ 25I]T4 added exogenously might in the liver. Also of interest is the low T3 and high explain this failure, but this is unlikely because incuba- rT3 serum concentration in patients with uncontrolled tion with ['251]T4 in serum-containing medium for 48 h diabetes mellitus in spite of severe hyperglycemia (39). gave the same results. Another possibility is that the Glucagon is reported to cause low T3 and high rT3 levels supraphysiological concentration of glucocorticoid in in the rat (40), although our in vitro data showed no sig- the culture medium, which is known to increase rT3 nificant inhibitory effect on 5'-deiodination in the ab- and decrease T3 concentration in humans (49), might sence of insulin. It is of interest to note that the low be responsible. This is also unlikely since hepatocytes insulin/glucagon ratio in catabolic states such as infec- grown in hydrocortisone-free Arg(-)DV medium sup- tion, traumatic and thermal injury, severe illness, and plemented only with insulin for 1-3 d showed rT3 but exercise (41) is also associated with decreased T3 and little T3 either in the medium or in the cells (unpub- increased rT3 in serum (42, 43). lished observations). The most likely explanation is that Insulin is a multipotent hormone that influences vari- proliferating liver cells are no longer normal cells and ous metabolic functions of most tissues (44, 45). The ef- metabolize T3 more rapidly than normal. The present fects may be rapid (within seconds to minutes, such data using proliferating hepatocytes (50) are reminis- as on membrane permeability), intermediate (within cent of the "low T3 syndrome" observed in the regener- hours, such as on protein synthesis) or delayed (within ating liver of partially hepatectomized rats (51). Fur- days, such as on RNA and DNA synthesis). From this thermore, ontogenetic changes in thyroid hormone point of view, the greater stimulatory effect of insulin metabolism were recently reported in immature observed after a 5-h incubation than after a 1-h incuba- chicken liver in which T4 deiodination mainly traverses tion suggests that the effect is an intracellular event, the non-T3-forming (probably rT3-forming) pathway such as on de novo enzyme synthesis, on inhibition of (52). Generally, various quantitative as well as quali- enzyme degradation, or on activation and inactivation tative alterations in enzymes occur during cell pro- of enzymes. The enzymes modulated by insulin may be liferation. In this culture method, 50% of cells in the the deiodinases themselves, enzymes involved in logarithmic stage are in S phase (50), and we have carbohydrate metabolism, or both. It should be pointed demonstrated in synchronized monkey hepatocar- out that the failure of Balsam and Ingbar (15) and of Jen- cinoma cells that deiodinases are synthesized pe-

Thyroid Hormone Metabolism 481 riodically with the highest activity in the late G, phase tion of thyroxine to triiodothyronine. Science (Wash. D. of the cell cycle (22). Our preliminary data suggest C.). 199: 904-906. 9. Chopra, I. J. 1980. Alterations in monodeiodination of that rT3 degrading enzymes are steadily decreas- iodothyronines in the fasting rat: effects of reduced ing in the cultured cells (unpublished observation), nonprotein sulfhydryl groups and hypothyroidism. Clin. which accounts at least in part for the accumulation of Endocrinol. Metab. 29: 161-167. rT3 in the medium. In view of the fact that some en- 10. Kaplan, M. M. 1979. Subcellular alterations causing re- zymes undergo alteration from adult to fetal type in duced hepatic thyroxine 5'-monodeiodinase activity in fasted rats. Endocrinology. 104: 58-64. primary cultured hepatocytes (53, 54), phenotypic 11. Harris, A. R. C., S. L. Fang, L. Hinerfeld, L. E. Braverman, changes might also occur in the deiodinases in primary and A. G. Vagenakis. 1979. The role of sulfhydryl groups cultured rat hepatocytes as seen in monkey hepatocar- on the impaired hepatic 3',3,5-triiodothyronine genera- cinoma cells (22). Although one of the greatest disad- tion from thyroxine in the hypothyroid, starved, fetal, and neonatal rodent.J. Clin. Invest. 63: 516-524. vantages of the primary culture of adult rat hepatocytes 12. Balsam, A., S. H. Ingbar, and F. Sexton. 1979. Observa- is the difficulty in obtaining nonproliferating hepato- tions on the factors that control the generation of tri- cytes for a number of days, methods have been reported iodothyronine from thyroxine in rat liver and the nature of extending the cell survival for up to 10-14 d (54). We the defect induced by fasting. J. Clin. Invest. 63: are extending our research to learn 1145-1156. whether this will 13. Gavin, L. A., F. Bui, F. McMahon, and R. R. Cavalieri. provide a more suitable model to investigate the regula- 1980. Sequential deiodination of thyroxine to 3,3'-di- tory mechanism of thyroid hormone metabolism in iodothyronine via 3,5,3'-triiodothyronine and 3,3',5'-tri- the liver. iodothyronine in rat liver homogenate. The effects of fasting versus glucose feeding.J. Biol. Chem. 254: 49-54. 14. Jennings, A. S., D. C. Ferguson, and R. D. Utiger. 1979. Regulation of the conversion of thyroxine to triiodo- ACKNOWLEDGMENTS thyronine in the perfused rat liver. J. Clin. Invest. 64: The authors thank Dr. Hans J. Cahnmann for assistance in the 1614-1623. synthesis of inner-ring labeled iodothyronines, Ms. Marcia 15. Balsam, A., and S. H. Ingbar. 1978. The influence of fast- Phyillaier for technical assistance, and Ms. Joan Todd and Ms. ing, diabetes, and several pharmacological agents on the Charlette Chun for the preparation of the manuscript. pathways of thyroxine metabolism in rat liver. J. Clin. Invest. 62: 415-424. 16. Leffert, H. L., K. S. Koch, T. Moran, and M. Williams. 1979. Liver cells. Methods Enzymol. 58: 536-544. REFERENCES 17. Sato, K., and J. Robbins. 1981. Glutathione deficiency 1. Vagenakis, A. G., A. Burger, G. I. Portnay, M. Rudolph, J. induced by cystine and/or methionine deprivation does T. O'Brian, F. Azizi, R. A. Arky, P. Nicod, S. H. Ingbar, and not affect thyroid hormone deiodination in cultured rat L. E. Braverman. 1975. Diversion of peripheral thyroxine hepatocytes and monkey hepatocarcinoma cells. Endo- metabolism from activating to inactivating pathways crinology. In press. during complete fasting. J. Clin. Endocrinol. Metab. 41: 18. Parman, U. A. 1979. Insulin. In Hormones in Blood. C. H. 191-194. Gray and V. H. T. James, editors. Academic Press, Inc., 2. Visser, T. J., S. W. J. Lamberts, J. H. P. Wilson, R. Docter, New York. 1: 55-170. and G. Hennemann. 1978. Serum thyroid hormone con- 19. Sorimachi, K., and H. J. Cahnmann. 1977. A simple syn- centration during prolonged reduction of dietary intake. thesis of [3,5-1251]diiodo-L-thyronine and of [3,5-1251]L- Clin. Endocrinol. Metab. 27: 405-409. thyroxine of high specific activity. Endocrinology. 101: 3. Spaulding, S. W., I. J. Chopra, R. S. Sherwin, and S. S. 1276-1280. Lyall. 1976. Effect of caloric restriction and dietary 20. Sato, K., and H. J. Cahnmann. 1980. Synthesis of [3,5- composition on T3 and reverse T3 in man. J. Clin. 125I]triiodo-L-thyronine of high specific activity. Anal. Endocrinol. Metab. 42: 197-200. Biochem. 102: 237-242. 4. Glass, A. R., R. Mellitt, K. D. Burman, L. Wartofsky, and R. 21. Leffert, H. L., and D. Paul. 1972. Studies on primary S. Swerdloff. 1978. Serum triiodothyronine in under- cultures of differentiated fetal liver cells. J. Cell Biol. 52: nourished rats: dependence on dietary composition rather 559-568. than total caloric or protein intake. J. Clin. Endocrinol. 22. Sato, K., and J. Robbins. 1980. Thyroid hormone metabo- Metab. 102: 1925-1928. lism in cultured monkey hepatocarcinoma cells: mono- 5. Suda, A. K., C. S. Pittman, T. Shimizu, and J. B. Chambers. deiodination activity in relation to cell growth. J. Biol. 1978. The production and metabolism of 3,5,3'-triiodo- Chem. 255: 7347-7352. thyronine and 3,3',5'-triiodothyronine in normal and 23. Flock, E. V., and J. L. Bollman. 1955. The metabolism of fasting subjects. J. Clin. Endocrinol. Metab. 47: 1311- thyroxine and triiodo-L-thyronine in the eviscerated rat.J. 1319. Biol. Chem. 214: 709-721. 6. Harris, A. R. C., S.-L Fang, F. Azizi, L. Lipworth, A. G. 24. Refetoff, S., N. I. Robin, and V. S. Fang. 1970. Parameters Vagenakis, and L. E. Braverman. 1978. Effect of starvation of thyroid function in serum of 16 selected vertebrates. on hypothalamic-pituitary-thyroid function in the rat. Endocrinology. 86: 793-805. Clin. Endocrinol. Metab. 27: 1074-1083. 25. Galton, V. A. 1971. Thyroxine metabolism and thyroid 7. Oppenheimer, J. H., and H. L. Schwartz. 1980. Factors function in rats following administration of estrogen. determining the level of activity of 3,5,3'-triiodothyronine- Endocrinology. 88: 976-982. responsive hepatic enzymes in the starved rat. Endo- 26. Seglen, P. 0. 1976. Preparation of isolated rat liver cells. crinology. 107: 1460-1468. Methods Cell Biol. 13: 29-83. 8. Chopra, I. J. 1978. Sulfhydryl groups and monodeiodina- 27. Roche, J., 0. Michel, R. Michel, and J. Tata. 1954. Sur

482 K. Sato and J. Robbins l'elimination biliaire de la triiodothyronine et de la L. J. DeGroot, G. H. Cahill, Jr., W. D. Odell, L. Martini, J. thyroxine et sur leur glycuroconjugaison hepatique. T. Potts, D. H. Nelson, E. Steinberger, and A. I. Winegrad, Biochim. Biophys. Acta. 13: 471-479. editors. Grune & Stratton Inc., New York. 2: 959-980. 28. Sekura, R. D., and W. B. Jacoby. 1979. Phenol sulfotrans- 42. Chopra, I. J., D. H. Solomon, U. Chopra, S. Y. Wu, D. A. ferase.J. Biol. Chem. 254: 5658-5663. Fisher, and Y. Nakamura. 1978. Pathway of metabolism of 29. Sekura, R. D., K. Sato, H. J. Cahnmann, J. Robbins, and W. thyroid hormones. Recent Prog. Hormone Res. 34: B. Jakoby. 1981. Sulfate transfer to thyroid hormones and 521-567. their analogs by hepatic aryl sulfotransferase. Enido- 43. O'Connell, M., D. C. Robbins, E. S. Horton, E. A. H. Sims, cri1nology. 108: 454-456. and E. Danforth, Jr. 1979. Changes in serum concentra- 30. Singer, T. P. 1975. Oxidative metabolism of cysteine and tions of 3,3',5'-triiodothyronine and 3,5,3'-triiodothy- cystine in animal tissues. In Metabolic Pathways. VII. ronine during prolonged moderate exercise. J. Clin. Metabolism of Sulfur Compounds. D. M. Green, editor. Endocrinol. Metab. 49: 242-246. Academic Press, Inc., New York. 535-546. 44. Pilkis, S. J., and C. R. Park. 1974. Mechanism of action of 31. Dodgson, K. S., and F. A. Rose. 1975. Sulfohydrolases. In insulin. Annu. Rev. Pharmacol. 14: 365-388. Metabolic Pathways. VII. Metabolism of Sulfur Com- 45. Goldfine, I. D. 1978. Insulin receptors and the site of ac- pounds. D. M. Green, editor. Academic Press, Inc., New tion of insulin. Life Sci. 23: 2639-2648. York. 359-431. 46. Richman, R. A., T. H. Claus, S. J. Pilkis, and D. L. Fried- 32. Van Middlesworth, L. 1974. Metabolism and excretion of man. 1976. Hormonal stimulation of DNA synthesis in thyroid hormones. Handb. Physiol. 3: 215-231. primary culture of adult rat hepatocytes. Proc. Natl. Acad. 33. Chopra, I. J. 1977. A study of extrathyroidal conversion of Sci. U. S. A. 73: 3589-3593. thyroxine (T4) to 3,3',5-triiodothyronine (T3) in vitro. 47. Armato, U., E. Draghi, and P. G. Andreis. 1978. Effect of Endocrinology. 101: 453-463. glucagon and insulin on the growth of neonatal rat 34. Herrera, E. F., F. Escobar Del Rey, and G. Morreale De hepatocytes in primary tissue culture. Endocrinology. Escobar. 1963. Effect of propylthiouracil on the in vivo 102: 1155-1166. deiodination of thyroxine labeled with 1311 in different 48. Bucher, N. L. R., and M. N. Swaffield. 1975. Regulation of positions. Endocrinology. 73: 744-747. hepatic regeneration in rats by synergistic action of 35. Visser, T. J., D. Fekkes, R. Docter, and G. Hennemann. insulin and glucagon. Proc. Natl. Acad. Sci. U. S. A. 72: 1978. Sequential deiodination of thyroxine by rat liver 1157-1160. homogenate. Biochem. J. 174: 221-229. 49. Chopra, I. J., D. E. Williams, J. Orgiazzi, and D. H. Solo- 36. Westgren, U., A. Melander, E. Wahlin, and J. Lindgren. mon. 1975. Opposite effects of dexamethasone on serum 1977. Divergent effects of 6-propylthiouracil on 3,5,3'- concentration of 3,3',5'-triiodothyronine, and 3,3',5-triiodo- triiodothyronine (T3) and 3,3',5'-triiodothyronine (RT3) thyronine.J. Clin. Endocrinol. Metab. 41: 911-920. serum levels in man. Acta Endocrinol. 85: 345-350. 50. Leffert, H. L., T. Moran, R. Boorstein, and K. S. Koch. 37. Flock, E., and J. L. Bollman. 1963. The effect of thiouracil 1977. Procarcinogen activation and hormonal control of on the metabolism of L-thyroxine. Biochem. J. 84: cell proliferation in differentiated primary adult rat liver 621-626. cell cultures. Nature (Lond.). 267: 58-61. 38. Sitar, D. S., and G. B. Hunninghake. 1975. Pharmaco- 51. Leffert, H. L., and N. M. Alexander. 1976. Thyroid hor- kinetics of propylthiouracil in man after a single oral dose. mone metabolism during liver regeneration in rats. J. Clin. Endocrinol. Metab. 40: 26-29. Endocrinology. 98: 1241-1247. 39. Kabidi, U. M., and B. N. Premachandra. 1980. Decreased 52. Borges, M., J. LaBourene, and S. H. Ingbar. 1979. Ontog- serum 3,5,3'-triiodothyronine (T3) and increased serum eny of peripheral iodothyronine metabolism in chick 3,5',3'-triiodothyronine (rT3) in uncontrolled diabetes embryo liver. 61st Annual Meeting of The Endocrine mellitus. 62nd Annual Meeting ofThe Endocrine Society. Society. Abstract. 237. Abstract. 214. 53. Leffert, H., T. Moran, S. Sell, H. Skelly, K. Ibsen, M. 40. Clarke, C. C., H. H. Bode, and P. A. Meara. 1979. The ef- Mueller, and I. Arias. 1978. Growth state-dependent fect of glucagon on T4 deiodination in vivo and in vitro. phenotypes of adult hepatocytes in primary monolayer 61st Annual Meeting of The Endocrine Society. Abstract. culture. Proc. Natl. Acad. Sci. U. S. A. 75: 1834-1838. 91. 54. Sirica, A. E., W. Richards, Y. Tsukada, C. A. Sattler, and 41. Unger, R. H., and L. Orci. 1979. Glucagon: secretion, H. C. Pitot. 1979. Fetal phenotypic expression by adult transport, and metabolism, physiologic regulation of se- hepatocytes on collagen gel/nylon meshes. Proc. Natl. cretion and derangements in diabetes. In Endocrinology. Acad. Sci. U. S. A. 76: 283-287.

Thyroid Hormone Metabolism 483