Extrathyroidal thyroxine-5′-monodeiodinase activity in cattle

Extrathyroidal thyroxine-5′-monodeiodinase activity in cattle

DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 1(4):279-290, 1984 EXTRATHYROIDAL THYROXINE-S-MONODEIODINASE ACTIVITY IN CATTLE S.Kahll, J. Bitmat- and T.S. R...

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DOMESTIC

ANIMAL

ENDOCRINOLOGY

Vol. 1(4):279-290,

1984

EXTRATHYROIDAL THYROXINE-S-MONODEIODINASE ACTIVITY IN CATTLE S.Kahll, J. Bitmat- and T.S. Rumsey3 Animal Science Institute, US Department of Agriculture, Agricultural Research Service, Beltsville, Maryland 20705 Received

April

5, 1984

ABSTRACT The monodeiodination of thyroxine (T,) to triiodothyronine (T,) was studied in vitro using liver, kidney, and muscle obtained from two-year old Angus and Hereford steers. Tissues were homogenized in .l M phosphate buffer-.25 M sucrose - 5 mM EDTA, pH 7.5, and centrifuged at 2000 X g for 30 min. Supernatants were incubated with T, (1.3 PM) at 37 C and T, generated was measured by radioimmunoassay of an ethanol extract of the incubation mixture. The T, to T, conversion in Angus liver homogenate was dependent upon pH, temperature, duration of incubation (5-120 min), homogenate (.025-.20 g-eq tissue/ml), and substrate concentration (.32-6.43 PM T,). The apparent K, and V,, of the conversion were .64 f.tM T, and 1.87 ng T, generated/hr/mg protein, respectively. Mean T, to T, conversion in Angus liver and kidney was 1.37 and .22 ng T,/hr/mg protein. The presence of 2 mM dithiothreitol (DTT), a sulfhydryl protective agent, significantly increased T, generation in liver and kidney (5.12 and 4.58 ng/hr/ mg protein) and also revealed activity in muscle (.05 ng/hr/mg protein). In liver and kidney from Hereford steers conversion activity was 2.89 and .48 in absence and 10.91 and 5.38 ng T,/hr/mg protein in presence of DTT, respectively. These results demonstrate the presence of a very active enzymatic system responsible for the peripheral 5’monodeiodination of T, to T, in cattle. INTRODUCTION It is well established that the only source of thyroxine (T4) in animal circulation and tissues is thyroidal secretion, while other iodothyronines are mainly generated in peripheral tissues during sequential deiodination of T, (for review see 1,2). The most potent thyroid hormone 3,3’,5-triiodothyronine (T,) is produced peripherally by 5’-monodeiodination of the phenolic ring of T,, and T, may be regarded as a prohormone. In contrast, 5-monodeiodination of the tyrosyl ring of T, results in the production of 3,3’,5’-triiodothyronine (rT,) which has no appreciable effect on metabolism (3). Extrathyroidal conversion of T, to T, has been demonstrated in in vivo studies in man (4,5), rat (6) and sheep (7). Also, in vitro techniques have been developed to measure T, deiodination directly in peripheral tissues and to study factors involved in T, to T, conversion. The 5’-deiodination has been demonstrated in various extrathyroidal tissues of man (8,9), rat (lo), mouse (1 l), dog (12), sheep (13), and pig (14). The T, to T, conversion was found to be enzymatic in nature and much more active in liver and kidney than in other tissues (10,15). Regulation of T, to T, conversion activity in peripheral tissues may be an important control point with respect to thyroid hormone action, serving as an adaptative mechanism in various physiological situations such as changes in food intake (14,15,16,17), ambient temperature variations (18,19), and during Copyright@

1994 by OOMENDO.

Inc.

279

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KAHL, BITMAN AND RUMSEY the neonatal period (13,20). Recently, decreased serum concentration resulting from decreased peripheral 5’-deiodination of T,, was reported linked dwarf chickens (2 1).

of T,, in sex-

There is no information concerning the peripheral monodeiodination of T, to T, in cattle. Furthermore, the pattern of T, and T, changes in plasma concentration (increase in T,, decrease in T3) found recently in our growth trials in steers after the application of hormonal (22,23) and non-hormonal (24) growth stimulators, suggested a relationship between growth rate and T, to T, conversion. The purpose of this study was to document and characterize T, to T, conversion activity in cattle tissues using an in vitro method. MATERIALS

AND METHODS

Preparation of homogenates. Tissues (liver, kidney and biceps femoris muscle) were obtained from two-year old Angus and Hereford steers. After an overnight fast the animals were killed, tissues removed, chilled on ice and transported to the laboratory for analysis within l-3 h. A 6 g portion of tissue was homogenized in 3 vol (wt/vol) ice cold .l M phosphate buffer, pH 7.5, containing .25 M-sucrose and 5 mM-EDTA using an Ultra Turrax homogenizer (Ika Werke, West Germany). The muscle tissue was homogenized in 5 vol of buffer. After centrifugation at 2000 X g for 30 min at 4 C, the supernatant (referred to hereafter as homogenate) was used for subsequent incubations. The homogenate was kept on ice if used immediately or frozen at -20 C for 1 or 2 days (no detectable decrease in deiodinating activity). Protein concentrations of homogenates were determined using bovine serum albumin (BSA) as a standard (25). Incubation procedure. Homogenates were incubated immediately after .preparation with T, for 30 min (except when time dependency was studied) in a shaking water bath at 37 C. The amount of T, generated was measured by specific radioimmunoassay of an ethanol extract. Incubations were performed in duplicate in 10 X 75 mm disposable glass culture tubes open to the air. The incubation mixture consisted of assay buffer (same as for homogenization) to adjust the incubation volume to 1 ml, .4 ml tissue homogenate and .l ml of T4 solution containing 1 ug of T, in .25% BSA in assay buffer. Final T, concentration was 1.3 l.tM except when enzyme kinetics were studied. The T, deiodination was stopped by placing the tubes in ice water, adding 2 ml 100% ethanol, and mixing. Samples were left in the freezer overnight (-20 C) and then centrifuged for 20 min at 3000 X g. Supernatants were kept at -20 C until T, was determined. Each assay included a control tube (blank) in which T4 was added at the end of incubation. The amount of T, in control tubes (. l.3 ng/mg protein) was subtracted from T, in test samples. Results were expressed as ng T, generated per mg tissue protein per 1 hr of incubation, except when specifically indicated. Recovery of stable T, (2-48 ng) in the ethanol extraction procedure was 101.7 -t 5.9% (mean + SD, n = 10) and recovery of i251labeled T, was 96.9 f 3.8%; therefore, data were not corrected for recovery. T, Radioimmunoassay. The amount of T, produced during incubation of T, with tissue homogenates was measured directly in the ethanol extract of the incubation mixture using Immuchem Covalent Coat RIA kit (Immuchem

EXTRATHYROIDAL

DEIODINASE ACTIVITY

281

Corp., Carson, CA) with the following modifications: the standards were prepared in buffer and ethanol was added to insure comparability to unknowns. 25-50 ~1 ethanol extract representing 8.3-16.7 u1 incubation mixture were added directly to the RIA tubes in which the final volume was 2.15 ml. The sensitivity of the assay was 8-17 pg per tube and cross-reactivity with T, and rT, was ,002 and .0004, respectively. Dilution curves of samples were parallel to the standard curve. Mean intra- and inter-assay coefficients of variations were 4.9 and 6.6%, respectively. Factors influencing in vitro T, generation by Angus liver homogenates. Incubation time was studied at 5, 15, 30, 60 and 120 min. Temperature was studied by incubating liver homogenates at 4, 25, 37 and 50 C. Enzyme concentration was evaluated using increasing amounts of liver homogenate (. 1 - .8 ml). To study pH, liver was divided into aliquots and homogenized in 4 buffer systems: .l M Tris-HCI (pH 8.0~9.0), .l M glycine-NaOH (pH 8.6-l 1 .O), .l M phosphate (pH 6.3-7.8), and .l M acetate (pH 3.6-5.6). Each buffer contained .25 M sucrose and 5 mM EDTA. The buffer for incubation was the same one used for homogenization. Substrate concentration was studied by incubating the liver homogenates in six T, concentrations from .32 to 6.43 PM. The effect of dithiothreitol (DTT) was determined by replacing .l ml of buffer with a .l ml buffer containing DTT to make a final concentration of 2 mM. Reagents. L-T,, L-T,, and dithiothreitol (DTT) were obtained from Sigma Chemical Company, St. Louis, Missouri. The iodothyronines were disolved in .l M NaOH and diluted with .25% BSA in incubation buffer. Statistical methods. Mean values of T, to T, conversion in respective tissues from Hereford and Angus steers were compared using Student’s t-test. RESULTS Figure 1 shows T, production as a function of time when T, was incubated with liver homogenate. The rate of T, generation was nearly linear during the first 60 min only with DTT (upper panel), and then declined. At each incubation time T, generation was several times higher in the presence of DTT. However, the ratio of T, produced in the presence of DTT to the T, produced without DTT increased from 2.56 at 5 min to 8.33 at 120 min of incubation. Net production of T, in 120 min was 15.02 rig/ml without DTT and 124.6 rig/ml with DTT, representing conversion of 1.5 and 12.4%, respectively, of the initial amount of T, present. No changes in homogenate T, concentrations over 120 min were observed in control tubes (3.68 and 4.05 rig/tube after 5 min and 3.45 and 4.44 rig/tube after 120 min without and with DTT, respectively). The 5’-monodeiodination of T, was optimal at pH 6.3 and 6.8 in the presence and absence of DTT, respectively (Figure 2). However, for all further studies pH 7.5 was chosen. An approximately 3 to !&fold enhancement of T, generation was found in the presence of 2 mM DTT at each pH studied. Maximal T, to T, conversion occurred at 37C and decreased at 25 or 50 C (Table 1). Production of T, was nearly completely abolished in homogenates placed in a boiling water bath for five minutes before incubation at 37 C. A linear relation was found between concentration) and T, to T, conversion 3). Despite a several-fold enhancement

homogenate concentration (enzyme only in the absence of DTT (Figure of T, to T, conversion by DTT, the

282

KAHL, BITMAN AND RUMSEY

+DTT

.4

.2

15

5

I

15

I

1

I

30

60

120

DURATION

OF

INCUBATION

(min)

Figure 1. Effect of duration of incubation (37 C and 1.3 phi initial T, concentration) on the conversion of T, to T, by steer liver homogenate in the absence (lower panel) and presence (upper panel) of 2 mM DTT. Each point represents the mean of two single incubations of the same homogenate preparation.

same rate of reaction was observed with the lowest (. 1 ml/tube) (.8 ml/tube) homogenate concentrations.

and highest

The effect of substrate (T4) concentration on the rate of T, generation is presented in Figure 4. Without DTT in the incubation tube (lower panel), T, production increased toward a maximum as the initial T, concentration increased to 4 PM. These data were examined by a double reciprocal plot of T, generation versus substrate concentration according to Lineweaver and Burk and a linear plot was obtained (Figure 5). The line of best fit using the least squares method yielded an apparent K, for T, of .64 PM and V,, of 1.87 ng/ hr/mg protein.

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283

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Effect of pH of the incubation mixture on the conversion in the absence (lower panel) and presence (upper panel) the mean of two single incubations of the same homogenate

10

I 11

of T, to T, by steer of 2mM DTT. Each preparation.

liver point

In another experiment (data not shown) apparent K, and V,, were .60 PM and 2.16 ng/hr/mg protein, respectively. In the presence of DTT a steady and progressive increase in T, generation was observed with increasing concentrations of T, (Fig. 4, upper panel). This curve did not show simple saturation kinetics and could not be linearized with either the Lineweaver-Burk or Hi11 plot. Therefore, the apparent K,,, of this process could not be estimated under this incubation regimen. TABLE 1. EFFECT OF TEMPERATURI HOMOGENATE. VALUES ARE MWS

OF INCUBATION ON THE CONVEFWON OF T, TO T, BY STEER LIVER + SD OF Two SEPARATE INCUBATIONS OF ME SAME HOMOGENATE PREPARATION.

Temperature C

T3 generation ng/hr/mg protein

4 ::

2

:3 * Before

incubation

the

homogenate

was

placed

in a boiling

water

.08 .40 1.04 .05 .I5

2 + f f -c

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bath

for

five

minutes

KAHL, BITMAN AND RUMSEY

284

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HOMOGENATE

(ml/incubation

Figure 3. Effect of tissue concentration on the conversion Each point represents mean of two separate incubations

I

.a

tube)

of T, to T, by steer liver homogenate. of the same homogenate preparation.

Table 2 compares the rate of T4 to T, conversion in liver, kidney and muscle homogenate preparations from Angus and Hereford steers. Liver tissue of Angus steers was found to be much more active than kidney in generating T, in the absence of DTT. No activity was detected in muscle preparations without DTT. The addition of DTT to the incubates enhanced the T, generation in liver (4fold increase), kidney (20-fold increase) and muscle homogenates. Both kidney and liver had approximately the same deiodination activity. In muscle tissue preparation, even with the presence of DTT, the rate of T, to T, conversion TABLE 2. COMPARISON

OF THE IN VITRO T, TO T, CONVERSION ACTIVITY FROM ANCL~S AND HEREFORD STEERS

Animal Breed

Tissue

Angus

Liver

no DTT 1.37 2 .20 .22

+

2.89

k

Muscle

. Values are duplicate. b Generation C Values are duplicate.

Kidney f SD of homogenates

not detectable. means f SD of homogenates

DTT

5.12

+

.78

.03

4.58

k

.28

.81

10.91

b

Liver mean

TISSCE HOMOGENATES

T, Generated ng/hr/mg protein

Kidney

Hereford

IN VARIOUS

from from

.48 two

.05

+ .I5 steers;

each

homogenate

10 steers;

each

homogenate

f

.Ol

+

2.10

5.38 2 .73 was incubated was

incubated

in in

EXTRATI-IYROIDAL

DEIODINASE ACTIVITY

T4

Figure 4. Effect absence (lower of two separate

CONCENTRATION

285

(I.IM)

of T, concentration on the T, to T, conversion by steer liver homogenate in the panel) and presence (upper panel) of 2 mM DTT. Each point represents the mean incubations of the same homogenate preparation.

was only ca 1% of that in liver. The same pattern in T, to T, conversion activity was found in tissue preparations from Hereford steers. However, T, generation was approximately 2-fold higher in Hereford liver and kidney homogenate without DTT (P<.O5) and in liver homogenate with DTT (P<.Ol) than in respective tissues from Angus steers. DISCUSSION In the present study we have demonstrated that homogenates of steer liver and kidney generate T, through a 5’-monodeiodination reaction of T, which is temperature, pH, time, tissue and substrate concentration dependent. These Endings support the presence of an enzymatic system responsible for T, to T, conversion in cattle tissues.

286

V =

KAHL, BITMAN AND RUMSEY

Tg

GENERATION

(ng/h/mg

protein)

.

K,,,: v max

1

1 1 /T4

Figure 5. Lineweaver-Burk point represents the mean

.64 :

UM 1.87

nglhlmg

2

protein

3

CUM)-’

plot of the T, to T, conversion of two separate incubations

activity by steer liver of the same homogenate

homogenate. preparation.

Each

The enzymatic nature of the 5’-monodeiodination was demonstrated by others in tissues of several animal species (10,12,15,26,27,28). Although the enzyme itself has not yet been isolated and purified, the 5’-deiodination activity was found to be located predominantly in microsomes and membrane rich fractions (29,30,31). However, cytosol or a source of reduced sulfhydryl groups such as reduced glutathione (GSH) or dithiothreitol (DTT) are required during T, to T, conversion (29,32). In the present study, a low speed postnuclear fraction of whole homogenate was used, and the T, to T, conversion activity showed simple saturation kinetics in the presence of increasing concentrations of substrate. However, apparent K, was calculated using the total amount of T, added to the incubation mixture and it does not reflect the real substrate concentration, i.e., free T, in the reaction system. Binding sites for T, were found in rat liver homogenate (33). Heinen et al. (28) reported that more than 99% of added T, was bound by the microsomal fraction in rat liver homogenates and the K,,, obtained using free T, as the substrate concentration was 200 times lower (9.7 nM T4) than the apparent K, calculated from added T, (2.1 uM T4). The values of apparent K, obtained in the present study for T, to T, conversion in steer liver homogenate are lower than values reported by others for rats (1.96 - 7.9 PM: 15,16,34), sheep (1.1 JAM: 13) and dog (16.7 FM: 12). This suggests that the enzymatic system responsible for T, to T, conversion might be more active at lower T, concentration in cattle tissues as compared to other species. However, differences in homogenate preparation and conditions of incubation could be responsible for the observed variation among the species.

EXTRATHYROIDAL

DEIODINASE ACTIVITY

The addition of 2 mM DTT, a sulfhydryl protective agent, increased the activity of the T, to T, conversion in liver and kidney and also made it possible to demonstrate this activity in muscle tissue homogenate (Table 2). The thiol dependence of 5’-monodeiodination is well established (29,32), and it was propsed (35) that thiol acts as a second substrate in the catalytic mechanism of T, to T, conversion. During the substrate (T4) conversion, an oxidized, inactive form of the enzyme is formed (sulfenyl iodide) and the thiol is involved in the subsequent reconversion of the modified enzyme to its native, active form by reducing its essential sulfhydryls (35). In addition, the study of Ozawa et al. (36) suggests that the DTT also interacts with T, making it more susceptible to 5’-monodeiodination. In the presence of DTT the T, to T, conversion reaction in our study did not follow Michaelis-Menten saturation kinetics. (Fig. 4, upper panel). In another experiment (data not shown) even with a substrate concentration as high as 26 uM T,, a steady increase in T, generation was found if DTT was present in the incubation system. One explanation of this relation could be a gradual saturation of T, binding proteins in homogenate, giving increases in the free fraction of T, at high T, concentration, while the active enzyme in the presence of DTT is not saturated. Our results are in close agreement with the observation of Kaplan (37) that during incubation of rat liver homogenate (2000 X g supernate fraction) with 1.3 -26 uM T, and 5 mM DTT, no saturating T, concentrations were achieved. As suggested by Kaplan (37) this was probably due to increases in both the apparent K, for T, and the V,, induced by DTT (38). Therefore, the addition of DTT increased the efficiency of the enzyme system in the whole tissue homogenate used in the present study. On the other hand, no relation was found between liver homogenate concentration and T, generation if DTT was present (Fig. 3). It is very difficult to find any correct explanation for this finding, since linear relationships (r2 = .99) occurred between these variables in the absence of DTT. Leonard and Rosenberg (31) reported that dilution of the whole rat kidney homogenate enhanced the T, to T, conversion activity and this phenomenon was attributed by Heinen et al. (28) to an increase in available free T, when less binding protein was added to the incubation system. Therefore, the constant rate of T, to T, conversion found in our study in the presence of DTT and increasing concentrations of homogenate may suggest that the addition of homogenate not only increased the concentration of the enzyme but also the concentration of binding protein, resulting in a decrease of free T, concentration available for deiodination. The sensitivity of the T, to T, conversion system to changes in T, concentration was much higher in the presence than in the absence of DTT (Fig. 4). The present results demonstrate differences in activity between steer liver and kidney homogenate (Table 2) in the absence of DTT. In the presence of DTT a much greater increase was observed in T, production in kidney than in liver homogenates. In rats, the animal species most commonly used for in vitro deiodination studies, the rate of T4 to T, conversion was nearly the same in liver and kidney, even without DTT (15) but much higher than in various other tissues (10). Activity of rat muscle homogenate was found to be only 5.2% of that in liver (10). In our study in steers no activity could be detected in muscle without DTT, and only 1.0% with DTT. This indicates that to study the Td to T, conversion in muscle, the separation and concentration of a 5’deiodinating rich tissue fraction is necessary.

KAHL, BITMAN

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In spite of the small number of animals used (especially Angus) the data of the present investigation revealed that the rate of T, generation was higher in Hereford than in Angus tissue homogenates. The conditions of assay were identical for both breeds but tissues were collected in December from Angus and in April from Hereford steers. Therefore, the observed differences may reflect the effect of breed, season, environmental temperature (19) and diet (17). In summary, this study indicates that in cattle the peripheral, extrathyroidal tissues participate in the production of an active form of the thyroid hormone. This aspect of thyroid physiology should be taken into consideration in the involvement of the thyroid hormones in growth, weight gain, milk production, and adaptation to environmental conditions. ACKNOWLEDGEMENTS

AND FOOTNOTES

I Visiting scientist from Academy of Agriculture, Krakow, Poland at University of Maryland, Department of Animal Sciences, College Park, Maryland 20742. 2 Milk Secretion and Mastitis Laboratory, USDA. To whom correspondence should be addressed. 3 Ruminant Nutrition Laboratory, USDA.

REFERENCES 1. Chopra IJ, Solomon DH, Chopra U, Wu SY, Fisher DA, Nakamura Y. Pathways of metabolism of thyroid hormones. Recent Prog Horm Res 34: 521-567, 1978. 2. Visser rJ. A tentative review of recent in vitro observations of the enzymatic deiodination of iodothyronines and its possible physiological implications. Mol Cell Endocrinol 10:241-247, 1978. 3. Pittman JA, Brown RW, Register HB. Biological activity of 3, 3’, 5’-triiodo-DLthyronine. Endocrinology 70:79-83, 1962 4. Braverman LE, Ingbar SH, Sterling K. Conversion of thyroxine (T,) to triiodothyronine (T,) in athyreotic human subjects. J Clin Invest 49:855-864, 1970. 5. Bianchi R, Mariani G, Molea N, VitekF, Cazzuola F. Capri A, Mazzuca N, Toni MG. Peripheral metabolism of thyroid hormones in man. I. Direct measurement of the conversion rate of thyroxine to 3,5,3’-triiodothyronine (T,) and determination of the peripheral and thyroidal production of T,. J Clin Endocrinol Metab 56: 1 l521163, 1983. 6. Schwartz HL, Surks MI, Oppenheimer JH. Quantitation of extrathyroidal conversion of L-thyroxine to 3,5,3’-triiodo-L-thyronine in the rat. J Clin Invest 50: 1124-l 130, 1971. 7. Fisher DA, Chopra IJ, Dussault JH. Extrathyroidal conversion of thyroxine to triiodothyronine in sheep. Endocrinology 9 1: 114 l- 1144, 1972. 8. Refetoff S, Matalon R, Bigazzi M. Metabolism of L-thyroxine (T,) and L-triiodothyronine (T,) by human fibroblasts in tissue culture: evidence for cellular binding proteins and conversion of T, to T,. Endocrinology 91:934-947, 1972. 9. Sterling K, Brenner MA, Saldanha VF. Conversion of thyroxine to triiodothyronine by cultured human cells. Science 179:1000-1001, 1973. 10. Chopra IJ. A study of extrathyroidal conversion of thyroxine (T,) to 3,3’,5-triiodothyronine (T,) in vitro. Endocrinology 101:453-463, 1977. 11. Harris ARC, Fang SL, Hinerfeld L, Braverman LE, Vagenakis AG. The role of sulfhydryl groups on the impaired hepatic 3’,3,5-triiodothyronine generation from thyroxine in the hypothyroid, starved, fetal, and neonatal rodent. J Clin Invest 63:516-524, 1979. 12. Lauberg P, Boye N. Outer and inner ring monodeiodination of thyroxine by dog thyroid and liver: a comparative study using a particulate cell fraction. Endocrinology 110:2124-2130, 1982. 13. Wu SY, Klein AH, Chopra IJ, Fisher DA. Alterations in tissue thyroxine-5’-monodeiodinating activity in perinatal period. Endocrinology 103:235-239, 1978. 14. Slebodzinski AB, Brzezinska-Slebodzinska E, Drews R. Reciprocal changes in serum 3,3’,5’-tri-iodothyronine concentration and the peripheral thyroxine inner ring monodeiodination during food restriction in the young pig. J Endocrinol 95:349355, 1982.

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