0013-7227/78/1033-0826$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society

Vol. 103, Nc. 3 Printed in U.S.A.

Metabolism of Thyroid Hormones by Rat Thyroid Tissue in Vitro* WILLIAM L. GREEN Robert H. Williams Laboratory for Clinical Investigation, Department of Medicine, Harborview Medical Center and University of Washington School of Medicine, Seattle, Washington 98104 ABSTRACT. Rat thyroid lobes or hemilobes have been incubated in Krebs-Ringer phosphate buffer containing labeled T4 and/or T3, and the products were separated by paper chromatography. Labeled T4 was actively degraded; about half of the T4 metabolized was recovered as T3. Labeled T3 was also metabolized, but less rapidly than T4. Other than T3 produced from T4, the major products from both hormones were inorganic iodide and iodoprotein; the latter was presumably a secondary product of iodide organification because its formation was inhibited by hypoxia and methimazole. Feeding the animals a low iodine diet increased their hormone-metabolizing activity. Incubation under nitrogen did not affect the rate of T4 degradation, but partially inhibited T3 degradation. Degradation of both hormones was unchanged in the presence of methimazole and ascorbate, was markedly inhibited by 1 mM

D

ESPITE earlier reports that ovine and canine thyroid slices did not deiodinate thyroid hormones (1, 2), it was later found that minces of thyroids derived from iodinedeficient, propylthiouracil-treated rats were able to degrade L-thyroxine producing 3,5,3'triiodo-L-thyronine (T3) as a major metabolite (3). Our interest in thyroidal iodothyronine deiodination stems from studies of intact human leukocytes in which it was shown that thyroid hormones are actively degraded during phagocytosis (4). Evidence was presented that degradation was in part mediated by a peroxidase, although nonperoxidative mechanisms seemed to be present (4-6). As thyroid cells and leukocytes have several properties in common, including the ability to iodinate proteins peroxidatively (7), the following studies were initiated to characterize the hormonemetabolizing system of rat thyroid tissue, to

Received September 19,1977. Address requests for reprints to: Dr. William L. Green, Harborview Medical Center, Department of Medicine, 325 Ninth Avenue, Seattle, Washington 98104. * This work was supported by USPHS Grant AM15810 from NIAMDD.

propylthiouracil (PTU), and was partially inhibited by azide and cyanide. Thyroid tissues concentrated both hormones, tissue to medium gradients averaging 5.4 for T4 and 20.7 for T3; none of the conditions affecting hormone degradation (incubation under nitrogen or with azide, cyanide, or PTU) significantly altered these gradients. It is concluded that the thyroid can metabolize both of its major hormones by a system distinct from thyroidal peroxidase. Hormone metabolism, therefore, is a potentially important factor in net hormone secretion. In its resistance to hypoxia, methimazole, and ascorbate and its sensitivity to PTU, the thyroid's system for generating T3 from T4 resembles T3-forming systems of liver and kidney. The thyroid, because T 3 formation is its dominant pathway for T4 metabolism, may provide a useful model for study of this reaction. (Endocrinology 103: 826, 1978)

compare it with the system in leukocytes, and to assess the role of peroxidases. A preliminary report of these findings was presented earlier (8). Materials and Methods Male Sprague-Dawley rats, weighing 150-500 g, were employed in all experiments. They received food, either Purina laboratory chow (NID) or commercial low iodine diet (LID) from Nutritional Biochemicals or General Biochemicals, and water ad libitum until about 1 h before sacrifice. Iodothyronines labeled in the phenolic ring were purchased from Industrial Nuclear, St. Louis, with specific activities of: [125I]T4 and [125I]T3, 100 juCi/jug; and [131I]T3, 80 juCi//ig. Methimazole (MMI) was a gift from Lilly Laboratories; other chemicals were obtained from commercial sources. The rats were killed by decapitation and the thyroids were quickly removed, trimmed of connective tissue, and placed in ice-cold Krebs-Ringeir phosphate buffer, pH 7.4 (KRP). Usually, each gland was divided into hemilobes and distributed to four incubation vessels. If this yielded too smalt an amount of tissue, hemilobes from additional glands were prepared and distributed to the same flasks, so that ultimately four flasks contained tis

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T4 AND T3 METABOLISM BY RAT THYROIDS sue from the same group of rats, allowing comparison of three treatments with control. In addition to these studies, in which the primary interest was the effect of various treatments in vitro on thyroid hormone metabolism, another series of experiments was performed in which the effect of various treatments in vivo on subsequent in vitro activity of thyroid tissue was assessed. For these studies, rats receiving NID were employed, and each lobe of the thyroid was incubated individually to provide duplicates from each animal. Only pooled control results from these latter studies were included in this paper; the animals reported here served as controls for studies of induced hypothyroidism, reported earlier (9), and of induced hyperthyroidism and starvation, to be reported later. The standard incubation medium was KRP containing human serum albumin (HSA; 0.05 mg/ml), [125I]T4 (3 juCi/ml), and [131I]T3 (1 fiCi/ml). In some instances, [125I]T4 or [125I]T3 was combined with Na131I in order to compare the fate of inorganic iodide with that of the selected iodothyronine. Test tubes (12 X 75 mm) containing 0.2 ml medium and varying amounts of tissue were incubated at 37 C under room air in a Dubnoff shaking incubator for 3 h. This incubation period was chosen after a preliminary experiment to define the time course of T4 breakdown and T3 formation suggested that these processes were approximately linear for 3 h. For incubations under N2 or O2, Warburg flasks containing 1.2 or 1.5 ml medium were gassed on the Warburg apparatus before tipping in labeled substrates. Incubations were terminated by adding 50 pi rat or human serum and sodium iodide to a final concentration of 30 I M and MMI to a final concentration of 20raM.Tissues were either homogenized in their own media or were blotted, weighed, and then homogenized in a small volume of KRP containing serum, Nal, and MMI. Aliquots of tissue and medium were then subjected to paper chromatography together with carrier iodide, T4) and T3, in two systems: butanol saturated with 2 N HAC (ascending; BA) and tertiary amyl alcohol saturated with 2 N ammonia (descending; TAA). Usually a third system, butanol-ethanol-0.5 N ammonia (5:1:2; ascending; BEA), was employed for confirmation of results. The paper chromatography strips were radioautographed, stained with ninhydrin, and cut into segments for counting in a dual channel automatic well counter, as previously described (10). The criterion for identifying compounds was the presence of a radioactive zone on the autograph corresponding precisely to the stained carrier. The results of chromatography in TAA were employed to estimate the proportion of radioactivity in var-

827

ious compounds. As T4 and iodide were often poorly separated in TAA, iodide was estimated from the results in BA, and was subtracted from the radioactivity in the combined T4 and iodide zones of TAA chromatograms to yield the proportion of T4. Overall T4 metabolism was estimated as: T4 lost =

-T4

x 100

where T4ini(inl is the proportion of T4 in medium from a tissue-free incubation and T4ruiiil is the proportion present after incubation with tissue. In order to determine the distribution of products, each metabolite was expressed as a proportion of the T4 lost. Thus, for T 3 formed from T4: T3 as % of products = ^

T 4:..:.:..,

~

rn

1 4.

When labeled T 3 was the starting material, similar formulae were employed to estimate the percentage of T3 lost and the distribution of products. To verify that the analytic methods did not cause artifactual breakdown of hormones in tissues, one experiment was conducted in which labeled T4 and T3 were added at the end of the incubation before homogenization. In this experiment, the distribution of radioactivity on paper chromatograms of tissue homogenates was identical to that of the starting material. In presenting results, the proportion of T4 metabolized is not corrected for tissue weight, as hormone metabolism was not linearly related to tissue weight (see Fig. 1). However, all comparisons are based on results of paired incubations employing similar amounts of tissue from the same rat or group of rats. When tissues and media were chromatographed separately, tissue/medium (T/M) gradients for hormones were computed as counts per min T4 (or T3)/mg tissue divided by counts per min T4 (or T3)/JU1 medium. In experiments with Na131I, organification of iodide was estimated from BEA or BA chromatograms, using a formula similar to that given above for the percentage of T4 lost, and T/M I" gradients were computed in a manner similar to that used for the hormones. Tests of significance included analysis of variance and the paired t test, performed according to Snedecor (11). In presenting control studies, the SD is given as an estimate of variability; SES are reported when comparing control results with results of various treatments. When analysis of variance was employed, the components of variance attributable to differences between different thyroid glands and to treatment effects were computed, and a variance ratio, F, was determined as variance due to treat-

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828

Endo • 1978 Vol 103 • No 3

ments divided by residual variance (11); the resid- hormone-binding materials from tissues, as ual variance was also employed to compute SE. discussed below; and 3) thyroids from LID

Results Results under standard incubation conditions Figure 1 depicts the overall rates of T4 and T 3 metabolism in a group of experiments with similar conditions, i.e. thyroid tissue was incubated at 37 C for 3 h in 0.2 ml medium containing small amounts of HSA and both labeled hormones, [125I]T4 and [131I]T3. Some rats received LID for 7-71 days (mean, 37 days), resulting in significant thyroid hypertrophy; mean thyroid weight ± SD was 10.2 ± 3.6 mg/100 g BW in the LID group, as opposed to 4.8 ±1.1 mg/100 g in the NID group. Mean body weight in the LID group (359 ± 68 g) was similar to that in the NID group (334 ± 72 g). Several features are apparent: 1) the proportion of T 3 metabolized was significant, but was always less than the proportion of T4 metabolized; 2) the amount of hormone metabolized was not directly proportionate to tissue weight, possibly due to the leakage of 30-

THYROIDS FROM LID RATS

I I T4

20-

1 T3

10-

(2)

(5)

(7)

(4)

(3)

5> J ^

0THYROIDS

!

30

?*

20-

"

&?

100-

NID

FROM

RATS

i

i

I

(7)

(3D

(ID

4.0-7.9 8.0-11.9 12.015.9

T3 (I)

(1)

16.019.9

20.023.9

24.027.9

Tissue weight (mg/flask)

FIG. 1. Metabolism of T4 and T3 by varying amounts of thyroid tissue from rats fed LID or NID. Figures in parentheses indicate number of individual incubations, all of which were conducted in 0.2 ml medium containing 3 ,iCi [125I]T4) 1 /iCi [131I]T3) and 0.05 mg HSA/ml. Values are given as mean ± SD. O, T4; • , T3. The upper panel depicts results with thyroid tissue from rats fed LID; the lower panel shows results from rats fed NID.

rats tended to metabolize T4 and T 3 more rapidly, although thyroids from NID rats did show significant hormone-metabolizing activity, in contrast to a prior report (3). Within the LID group, there was a significant correlation between length of LID feeding and the percentage of T4 lost (r = 0.56, P = 0.01). The correlation with the percentage of T 3 lost (r = 0.32) was not significant. Table 1 shows the chromatographically determined distribution of products of T4 and T 3 metabolism; the average composition of media incubated without tissue is also given. The production of origin material, presumed to be iodoprotein, and of iodide varied considerably, but the recoveries of these two metabolites were reciprocally related. Combining the experiments with LID and NID rats shown in Table 1, the correlation coefficients between production of origin material and production of iodide from [125I]T4 and from [I31I]T3 were —0.97 and —0.86, respectively. Thus, the variance of the sum, (O -I- I), in this series was much smaller than the variance of either component, e.g. (O + I) produced from T4 by thyroids from LID rats comprised 73.0 ± 4.6% (SD) of T4's products. Also, the production of origin material could be inhibited, with a corresponding increase in iodide, by methimazole and by hypoxia. Thus, it is likely that iodoprotein is a secondary product of iodothyronine metabolism, produced by organification of iodide released from T4 or T3. Small increments of radioactivity in the zone between T4 and T 3 on TAA chromatography, designated Xi in the table, were seen consistently after incubation with T4; the nature of this material is uncertain, as both tetraiodothyroacetic acid (Tetrac) and 3,3'-diiodo-L-thyronine (T2) migrate here (12). Besides iodide and iodoprotein, the other major product of T4 metabolism was T3, comprising about XA of the total products. The difference between T 3 formation by LID and NID thyroids was significant (P < 0.05). However, it is possible that the large]: amounts of T3-like radioactivity in the starting material for the NID experiments contributed to this difference. To assure that any conclu-

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T4 AND T 3 METABOLISM BY RAT THYROIDS

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TABLE 1. Results of control incubations with both [ I25 I]T 4 and [ i; "I]T :! Chromatography of tissue-free media" Diet

n

6

LID

9

NID

17

[

125

I]T4

I]T,

0

I

T4

X,

T3

Front

0

I

X2

T3

Triac

Front

1.5 (0.4) 1.5 (0.4)

1.5 (0.8) 2.0 (1.0)

94.2 (2.0) 93.7 (1.9)

1.2 (0.3) 1.2 (0.3)

0.8 (0.2) 0.9 (0.3)

0.8 (0.8) 0.7 (0.5)

1.0 (0.5) 0.8 (0.3)

3.4 (2.1) 2.6 (0.9)

7.4 (2.6) 6.3 (1.7)

85.6 (5.1) 87.4 (3.2)

1.6 (0.4) 1.5 (0.4)

1.0 (0.6) 1.2 (0.6)

Results of tissue incubations rl25

['"I]rp fv I A, 10

LID

9

NID

17

lost 25 .3 (5 .8) 19 .5 (4 .6)

I]T 4 , distribution of products 0

rl.il

[ K "I]-

I]T.t, distribution of products'

1.3, 10

0 34.3 (21.7) 21.1 (10.0)

I 38 (19 54 (9

X, .7 .7) .4 .9)

1.3 (1.1) 2.0 (0.8)

T3 25 (4 22 (2

.0 .0) .3 .3)

Front 0.6 (1.1) 0.2 (1.9)

lost 18 (7 12 (2

.4 .1) .4 .9)

0

I

66.9 (56.3) 34.9 (19.4)

50.2 (44.2) 83.1 (18.0)

X, -19 (23 -20 (13

.4 .8) .8 .1)

Triac

Front

2.3 (2.0) 3.9 (0.9)

0 (5 .7) - 1 .1 (2 .5)

Xi, Radioactivity on TAA chromatograms between the T 4 and T) zones; Xa, between the iodide and T.i zones; 0, from point of application to iodide zone. Triac, 3,5,3'-triiodothyroacetic acid. LID was given for an average of 37 days (range, 7-71 days). " Values are given as means percentage of total 125I or 131I in each zone; SD is in parentheses. 6 Number of experiments, each with one to four replicate flasks. c Values are given as change in radioactivity in each zone divided by change in amount of labeled T 4 or T.j times 100; SD is in parentheses.

sions concerning T3 formation were conservative, the amount of radioactivity in the T3 zone of tissue-free media was subtracted from the amount found after tissue incubations (see Materials and Methods). If, however, most of the "T3" radioactivity initially present was metabolized, T 3 production would be underestimated. In the extreme case, if all of the initial T3-like material disappeared, estimated T3 formation, as a percentage of T4's products, would be 28% and 27% for LID and NID thyroids, respectively. In any case, the differences are small enough that they are of doubtful biological significance. To determine whether the rate of T3 degradation was an important determinant of the relative amounts of T3 and iodide recovered after incubation with T4, correlation coefficients were computed between the percentage of [131I]T3 lost and [125I]T3 formed, expressing the latter as a percentage of the products of T4 metabolism. Data from the dual label experiments of Fig. 1 and Table 1 were employed. In the studies with LID rats, there was a significant negative correlation (r = —0.75, P < 0.01); in studies with NID rats, no significant correlation was found (r = —0.09).

One problem relative to the chromatographic procedure concerns 3,3',5'-triiodo-Lthyronine (rT3), which migrates close to T4 in TAA (12) and was not isolated in our system. Preliminary studies suggest that thyroid tissue rapidly degrades rT3, producing both iodide and radioactivity migrating with T2. Therefore, it is likely that most of any rT3 formed would be interpreted as deiodination of T4; some of it might lead to material in the Xi zone. The T 3 preparations employed contained large amounts of radiaoctivity in the "X2" zone (Table 1), which potentially includes rT3, T4, Tetrac, and T2 (12), rendering conclusions about this group of compounds uncertain. The rapid disappearance of some labile contaminant, such as T2, may overshadow the appearance of important T 3 metabolites in this zone. In addition to the experiments described above, a number of studies were conducted with media containing only one labeled iodothyronine; also, albumin-free media were sometimes employed. These variations produced no significant changes in metabolism of T4 or T3; therefore, in reporting effects of various agents on hormone metabolism below,

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GREEN

830

experiments with a single iodothyronine and with no albumin are pooled with those employing the conditions described in Fig. 1. Higher concentrations of albumin, employed in some early studies, also had little effect on hormone metabolism. A systematic study of alterations produced by varying albumin concentrations was not performed, but T/M gradients did seem to be lowered by media containing >0.5 mg HSA/ml. Study of T/M gradients was not the initial purpose of this study, and gradients were not determined in most of the studies of Fig. 1 and Table 1. The pooled results of incubations in which gradients were determined, including experiments with a single iodothyronine and with no albumin, are given in Table 2. It is

Endo Vol 103

1978 No 3

Warburg apparatus, larger amounts of tissue (25-75 mg) were incubated in 1.2-1.5 ml medium. All tissues were from rats on LID for 26-53 days. Mean values for total T4 and T 3 degradation are depicted in Fig. 4. Analysis of variance of the results indicated a significant

apparent that gradients for T 3 were much

greater than for T4. There is a suggestion of greater binding by tissue from NID rats; also a negative correlation with tissue weight, significant in the case of T4, was found. Effects of added stable T4 or T3 The proportion of labeled hormone degraded was not greatly altered by adding stable hormone until concentrations greater than 3 ]UM were reached (Figs. 2 and 3). At higher concentrations, T4 inhibited Ta's metabolism, and vice versa. Up to a concentration of 10 /xM, T/M gradients were not affected; indeed, stable T4 may have increased the gradient for labeled T4. These results imply a large capacity of thyroid tissue to accumulate and degrade both of its characteristic hormones. Incubations under iV2 and O2 In these experiments, conducted on the

0 0.5 1 2 4 10 Concentration of added T4 or T3 (MM) FIG. 2. Effect of stable T4 and T3 on metabolism of labeled T4. Tissues were obtained from rats fed LID for 20-50 days. Degradation of T4 is expressed as a percentage of control value, i.e. as a percentage of the value obtained with no added stable T4 or T3. In the controlflasks,the concentration of labeled T4 was 0.05 JUM and the mean value of ± SD for the percentage of T4 lost was 31.6 ± 10.7. In one experiment (•), T/M gradients were determined from a single flask at each concentration of T4 or T3. Otherwise, each symbol represents results ± SE from an individual experiment in which two or three replicate incubations were performed at each iodothyronine concentration. Where SE is not shown, it was less than the height of the symbol.

TABLE 2. T/M gradients

LID rats

NID rats

Total

Wt"

T/M

n

Wt

T/M

n

Wt

T/M

T4

9.5 (2.7)

7.6 (1.9)

12

17.0 (3.9)

4.1 (1.6)

19

14.3 (5.1)

5.4 (2.4)

-0.61"

T,

8.9 (2.8)

23.2 (6.5)

16.3 (4.2)

19.2 (2.3)

13

13.5 (5.2)

20.7 (4.6)

-0.42

n

SD is in parentheses. " Tissue weight, milligrams per flask; each flask contained 0.2 ml medium. * Correlation coefficient, tissue weight vs. T/M. c P In the studies with [12r>I]T4 and [i:ilI]T3> endogenously formed [125I]T3 could be degraded at quite a different rate than exogenous [I31I]T3, and to correct [125I]T3 recovery by employing data for [I31I]T3 breakdown would require more data than the present studies afford concerning the rates at which T3 from each source reaches T3-degrading sites, and the rate and extent of mixing before degradation. Assuming, however, that [I31I]T3 entering the tissue mixes completely with newly formed [125I]T3 before degradation and that the I25I:13II ratio in this well mixed intrathyroidal T 3 pool remains constant during incubation, then the ratio between tissue T 3 content and the amount of T3 degraded should be the same for both isotopes. In studies where tissue and medium were analyzed separately, [12r>I]T3 degraded is the only unknown in the expression which results from these assumptions:

[12r'I]T3 in tissue [K"I]T3 in tissue I25 [ I]T3 degraded ~ [131I]T3 degraded Adequate data are available from 10 of the incubations with LID thyroids shown in Fig. 1. In these studies, the mean actual recovery of [12r>I]T3 ± SD, expressed as a percentage of products, was 27.8 ± 3.7%, and corrected recovery was 35.3 ± 2.5%.

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836

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metabolism has been suggested by the ability of purified peroxidases to degrade iodothyronines (19) and by the presence of peroxidaselike hormone-degrading activity in homogenates of various rat tissues (20). In our study of phagocytosing leukocytes, parallelism between the hormone-deiodinating activity of purified myeloperoxidase and of intact cells, particularly their common responses to various inhibitors, made it seem likely that deiodination by intact leukocytes was in part mediated by peroxidase (4). It has also been shown that a peroxidase present in thyroid homogenates can degrade thyroid hormones in the presence of hydrogen peroxide (21). Our efforts to demonstrate peroxidative breakdown of T4 by thyroid tissue, however, were unsuccessful. Deiodination of T4 was not inhibited by hypoxia nor by ascorbate, conditions which inhibited both the myeloperoxidase system and phagocytosing leukocytes. MMI, which stimulated deiodination by myeloperoxidase and inhibited deiodination by other peroxidases (4), had only a minor effect in the thyroid. Partial inhibition of T4 deiodination could be produced by incubating thyroids with cyanide and azide, but the concentrations required greatly exceeded those which inhibited iodide organification, presumed to be a peroxidative process, and were even greater than those inhibiting the iodideconcentrating mechanism, which is thought to depend on phosphate bond energy (22). Although peroxidases and other oxygen-dependent systems may operate in leukocytes and elsewhere, they do not seem to be important mediators of T4 breakdown in the thyroid. Moreover, oxygen-dependent deiodination of T4 in leukocytes produces very little T3 (4, 5), another contrast with thyroidal deiodination. Breakdown of T3 was inhibited by hypoxia, affording some evidence that separate systems mediate T4 and T 3 metabolism. T3 metabolism may also have been more susceptible to inhibition by cyanide and azide. As with T4, however, MMI and ascorbate were ineffective and deiodination was only partially inhibited by high concentrations of cyanide and azide. Thus, it seems unlikely that a typical peroxidase is involved in thyroidal T3 metabolism;

Endo • 1978 Vol 103 • No 3

rather, an oxygen-dependent deiodinase, similar to the nonperoxidative system postulated in leukocytes (4-6), may be present. There is now abundant evidence that tissues other than the thyroid are capable of converting T4 to T3, and the question arises whether a similar enzymatic mechanism mediates the reaction in these various tissues. Inhibition of conversion is produced by PTU and similar compounds in vivo (23-25), as well as in kidney slices (26), kidney homogenates (27), liver slices (10), and mouse neuroblastoma cells (28); thyroid tissues share this property. We have presented evidence that T3 formation in the liver, as in the thyroid, is oxygen independent and is not inhibited by MMI or ascorbate (10); T3 formation by kidney slices is similar (29). Also, both liver and kidney slices resemble thyroid tissue in their responses to cyanide and azide, i.e. high concentrations of these agents cause partial inhibition of T3 formation (unpublished observations). Studies with kidney slices, performed over 2 decades ago, first demonstrated that production of T 3 from T4 is resistant to hypoxia (30) and inhibited by 10 mM cyanide (31). A more recent investigation has demonstrated such oxygen independence in cell-free preparations from rat kidney (27). It is possible, therefore:, that thyroidal T4 metabolism is mediated by a 5'-deiodinase similar or identical to the enzyme in other tissues. The high activity in the thyroid and the relative inactivity of competing pathways make it a convenient model for study of this reaction.

Acknowledgments The author thanks Ann Weinmann, Sondra Goehle, and David Roberson for expert technical assistance, and. Cheryl Tomyn for preparation of the manuscript.

References 1. Roche, J., R. Michel, O. Michel, and S. Lissitzky, Sur la deshalogenation enzymatique des iodotyrosines par le corps thyroide et sur son role physiologique, Biochim Biophys Ada 9: 161, 1952. 2. Tong, W., P. Kerkof, and I. L. Chaikoff, Iodine metabolism of dispersed thyroid cells obtained by trypsinization of sheep thyroid glands, Biochim Biophys Ada 60: 1, 1962. 3. Haibach, H., Evidence for a thyroxine deiodinating

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T4 AND T, METABOLISM BY RAT THYROIDS

4.

5.

6.

7. 8.

9.

10.

11. 12.

13. 14.

15.

16.

17.

18.

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