Relation of Triiodothyronine and Reverse Triiodothyronine Administration in Rats to Hepatic L-Triiodothyronine Aminotransferase Activity1 NORMAN FISHMAN,2 YEE PING HUANG, DORIAN C. TERGIS, AND RICHARD S. RIVLIN Department of Medicine and Institute of Human Nutrition, College of Physicians and Surgeons of Columbia University, New York, New York 10032 of 5 /xg/100 g BW for 8 days significantly elevated hepatic enzyme activity above normal. In daily doses of 2.5 fig/100 g BW, T3 restored the depressed enzyme activities in hypothyroid rats to normal. Daily administration of 3,3',5'-triiodothyronine (reverse T3) to normal rats in doses of 17.5 fig/ 100 g BW and greater for 3 days increased L-T3 aminotransferase activity more than 30% above normal levels. Reverse T3 appeared to be approximately as active as T3 in increasing the hepatic activity of L-T3 aminotransferase. (Endocrinology 100: 1055, 1977)
ABSTRACT. The effects of administration of 3,5,3'triiodothyronine (T3) to normal and to hypothyroid male rats upon the hepatic activity of L-triiodothyronine aminotransferase were determined using 3,5-dinitro-L-tyrosine as substrate in the assay. Initial studies in normal rats demonstrated that basal enzyme activity was highest in liver and kidney of the organs tested, and that virtually no activity was detectable in skeletal muscle, serum, thyroid or pituitary gland. Hepatic enzyme activity increased from birth to a peak at 80-120 days and declined thereafter. Daily administration of T3 to normal rats in doses
A
NEW enzyme (L-triiodothyronine aminotransferase) which catalyzes the transamination of thyroxine, triiodothyronine and substituted tyrosines was described several years ago in rabbit organs (1,2). This enzyme is located in the soluble fraction of the cell, and with pyruvate as substrate, exhibits specificity for T3 rather than acting upon both T3 and T4. L-Triiodothyronine aminotransferase differs in several important properties from another thyroid hormone transaminase which had been reported earlier in the rat (3). The latter enzyme is mitochondrial in location, utilizes L-thyroxine preferentially, and does not act upon 3,5-dinitro-L-tyrosine. Our initial investigations of L-triiodothyronine aminotransferase in rats indicated that it was under thyroid hormone control, increasing in activity after administration of either T3 or T4 and decreasing after thyReceived June 14, 1976. 1 These studies were supported by Grants AM 15265 and CA 12126 from U.S. Public Health Service and the Stella and Charles Guttman Foundation. 2 Present address: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
roidectomy (4,5). In an effort to learn more about the physiological role of this enzyme, measurements were made of its tissue distribution in the rat, its activity in relation to age, the time course of its response to T3 and the doses required, and finally its activity after administration of reverse T3. Materials and Methods Animals All experiments were performed using male white rats obtained from the Holtzman Company, Madison, Wisconsin. Solutions of T3 and of reverse T3 (3,3',5'-triiodo-D,L-thyronine) were administered by daily ip injection with the doses specified for every experiment. In each instance, age- and sex-matched controls received injections of isotonic saline of the same volume and pH as that of the hormone solutions. Serum levels of T 3 and of reverse T 3 were not determined in these studies. Animals were radiothyroidectomized by the administration of a single ip dose of 131I, 300 /iCi/ 100 g body weight, after they had fed on a lowiodine diet (General Biochemicals Corporation) for the preceding three to four weeks. Fortyeight hours after receiving 131I, these animals were switched to a diet of Purina Chow which
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1056
FISHMAN ETAL.
they received ad libitum throughout the experimental period. Animals were utilized in experiments a minimum of two months following treatment with 131I. Documentation of the production of hypothyroidism by this technique has been presented previously (6). All animals received a diet of rat Purina Chow fed ad libitur.i except as noted above. Reagents 3,5,3'-Triiodo-L-thyronine, was purchased from the Nutritional Biochernicals Corporation. Pyridoxal phosphate, potassium a-ketoglutarate and 3,5-dinitro-L-tyrosine were purchased from Sigma Corporation. Samples of 3,3',5'-triiodoD,L-thyronine (reverse D,L-T 3 , rT3) were generously provided by Dr. Robert I. Meltzer, WarnerLambert Research Institute. This product is estimated by the manufacturer to contain less than 1% T3. The effect of any contamination with this amount of T3 or other thyroid hormone intermediates would be minimal. Na131I, free from reducing agent, was purchased from Amersham/Searle Corporation, and diluted with isotonic saline for injection. Preparation of tissues Animals were sacrificed by decapitation and exsanguination. Tissue samples were removed promptly and placed on ice until homogenization. These samples were generally assayed fresh, but could be maintained in the frozen state (-20 C) for 2 months without appreciable loss of activity. Each specimen was homogenized in 2 volumes of a buffer containing 20 mM Tris-HCl (pH 7.8), 30 mM KC1, 5 mM magnesium chloride, 0.25M sucrose, 1 mM EDTA and 5 mM 2-mercaptoethanol. Homogenization was performed in a Teflon tissue grinder for a 2 min period. The resulting homogenates were passed through 2 layers of cheesecloth. An aliquot was removed for assay. Enzyme assay The activity of L-triiodothyronine aminotransferase was assayed by the procedure of Soffer and co-workers (1,2) which utilizes 3,5-dinitroL-tyrosine as substrate. The assay measures the rate of formation of the stable colored compound, 3,5-dinitro-p-hydroxyphenylpyruvic acid, spectrophotometrically as an enol-borate complex. The artificial substrate, 3,5-dinitro-L-tyro-
Endo • 1977 Vol 100 • No 4
sine, is used in place of L-triiodothyronine because of the simplicity and accuracy of the spectrophotometric assay, because it is more economical, and because transaminating activities for both compounds have been copurified during the entire procedure in which the enzyme has been isolated and purified (2). 3,5-Dinitro-Ltyrosine has zero activity relative to tyrosine as a substrate for tyrosine aminotransferase (7). The reaction mixtures of 0.9 ml contained 67 mM Tris-HCl (pH 7.8), 20 mM potassium aketoglutarate, 10 mM 3,5-dinitro-L-tyrosine, 10 mM 2-mercaptoethanol, 20 /iM pyridoxal phosphate, and aliquots of tissue homogenates. Generally, 2 fxl volumes of liver homogenates were used; larger aliquots were required for tissue samples with lower enzyme activity. The reaction mixtures were incubated for 60 min at 37 C, and the reactions stopped by the addition of 1.2 ml of 10% trichloroacetic acid. The precipitates were removed by centrifugation at 2000 x g for 10 min. Aliquots of 1.5 ml were removed from each supernatant solution and mixed with an equal volume of a solution containing 2M sodium arsenate and 1M sodium borate, pH 6.5. All tubes were incubated for 30 min at room temperature, and the concentration of the enol-borate complex of 3,5-dinitro-phydroxyphenylpyruvic acid was determined spectrophotometrically at 320 nm, using a molar extinction coefficient of 12,750 (2). Blank samples containing no enzyme were prepared and assayed similarly. One unit of enzyme activity is defined as that amount which results in the formation of 1 /xmol of 3,5-dinitro-p-hydroxyphenylpyruvic acid per min under standard assay conditions. In the assay the molar amounts of glutamic acid and 3,5dinitro-p-hydroxyphenylpyruvic acid formed are equivalent (2). Determination of protein was done by the method of Lowry et at. (8), in which a solution of bovine serum albumin was used as a standard. Each experiment was carried out in its entirety three or more times. Direct effects of thyroid hormones in vitro have not been observed other than as substrates for the enzyme.
Results
Distribution of L-triiodothyronine aminotransferase activity In order to determine the distribution of enzyme activity in the rat, measurements
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1057
HEPATIC T3 AMINOTRANSFERASE ACTIVITY
TABLE 1. Distribution of L-triiodothyronine aminotransferase activity in various rat organs; results are shown in samples pooled from 2 male rats aged 90 days
were made in various organs and tissues pooled from 2 male rats aged 90 days. As is shown in Table 1, the highest enzyme activity per mg protein was demonstrable in kidney, followed by liver. Enzyme activity in whole brain was less than 10% of that in kidney and liver. Lung, heart, testes and spleen had low but measurable enzyme activity. Virtually no activity was demonstrable in skeletal muscle, thyroid gland, pituitary gland or blood serum.
Hepatic L-triiodothyronine aminotransferase activity in relation to age Measurements were made of hepatic Ltriiodothyronine aminotransferase activity in rats ranging in age from several hours after birth to 290 days. As shown in Fig. 1, enzyme activity remains fairly stable for the first few weeks afterbirth. Thereafter, progressive increases in enzyme activity occur until an apparent peak is reached at 80-120 days of age. In rats older than 120 days, enzyme activity begins to decrease, and levels recorded at 290 days were not significantly greater than at birth.
Organ
L-triiodothyronine aminotransferase activity (milliunits/mg protein)
Kidney Liver Brain Heart Lung Testes Spleen Other organs*
40.5 17.7 0.121 0.050 0.043 0.031 0.016
ABSTRACT. The effects of administration of 3,5,3'triiodothyronine (T3) to normal and to hypothyroid male rats upon the hepatic activity of L-triiodothyronine aminotransferase were determined using 3,5-dinitro-L-tyrosine as substrate in the assay. Initial studies in normal rats demonstrated that basal enzyme activity was highest in liver and kidney of the organs tested, and that virtually no activity was detectable in skeletal muscle, serum, thyroid or pituitary gland. Hepatic enzyme activity increased from birth to a peak at 80-120 days and declined thereafter. Daily administration of T3 to normal rats in doses
A
NEW enzyme (L-triiodothyronine aminotransferase) which catalyzes the transamination of thyroxine, triiodothyronine and substituted tyrosines was described several years ago in rabbit organs (1,2). This enzyme is located in the soluble fraction of the cell, and with pyruvate as substrate, exhibits specificity for T3 rather than acting upon both T3 and T4. L-Triiodothyronine aminotransferase differs in several important properties from another thyroid hormone transaminase which had been reported earlier in the rat (3). The latter enzyme is mitochondrial in location, utilizes L-thyroxine preferentially, and does not act upon 3,5-dinitro-L-tyrosine. Our initial investigations of L-triiodothyronine aminotransferase in rats indicated that it was under thyroid hormone control, increasing in activity after administration of either T3 or T4 and decreasing after thyReceived June 14, 1976. 1 These studies were supported by Grants AM 15265 and CA 12126 from U.S. Public Health Service and the Stella and Charles Guttman Foundation. 2 Present address: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
roidectomy (4,5). In an effort to learn more about the physiological role of this enzyme, measurements were made of its tissue distribution in the rat, its activity in relation to age, the time course of its response to T3 and the doses required, and finally its activity after administration of reverse T3. Materials and Methods Animals All experiments were performed using male white rats obtained from the Holtzman Company, Madison, Wisconsin. Solutions of T3 and of reverse T3 (3,3',5'-triiodo-D,L-thyronine) were administered by daily ip injection with the doses specified for every experiment. In each instance, age- and sex-matched controls received injections of isotonic saline of the same volume and pH as that of the hormone solutions. Serum levels of T 3 and of reverse T 3 were not determined in these studies. Animals were radiothyroidectomized by the administration of a single ip dose of 131I, 300 /iCi/ 100 g body weight, after they had fed on a lowiodine diet (General Biochemicals Corporation) for the preceding three to four weeks. Fortyeight hours after receiving 131I, these animals were switched to a diet of Purina Chow which
1055 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 02:17 For personal use only. No other uses without permission. . All rights reserved.
1056
FISHMAN ETAL.
they received ad libitum throughout the experimental period. Animals were utilized in experiments a minimum of two months following treatment with 131I. Documentation of the production of hypothyroidism by this technique has been presented previously (6). All animals received a diet of rat Purina Chow fed ad libitur.i except as noted above. Reagents 3,5,3'-Triiodo-L-thyronine, was purchased from the Nutritional Biochernicals Corporation. Pyridoxal phosphate, potassium a-ketoglutarate and 3,5-dinitro-L-tyrosine were purchased from Sigma Corporation. Samples of 3,3',5'-triiodoD,L-thyronine (reverse D,L-T 3 , rT3) were generously provided by Dr. Robert I. Meltzer, WarnerLambert Research Institute. This product is estimated by the manufacturer to contain less than 1% T3. The effect of any contamination with this amount of T3 or other thyroid hormone intermediates would be minimal. Na131I, free from reducing agent, was purchased from Amersham/Searle Corporation, and diluted with isotonic saline for injection. Preparation of tissues Animals were sacrificed by decapitation and exsanguination. Tissue samples were removed promptly and placed on ice until homogenization. These samples were generally assayed fresh, but could be maintained in the frozen state (-20 C) for 2 months without appreciable loss of activity. Each specimen was homogenized in 2 volumes of a buffer containing 20 mM Tris-HCl (pH 7.8), 30 mM KC1, 5 mM magnesium chloride, 0.25M sucrose, 1 mM EDTA and 5 mM 2-mercaptoethanol. Homogenization was performed in a Teflon tissue grinder for a 2 min period. The resulting homogenates were passed through 2 layers of cheesecloth. An aliquot was removed for assay. Enzyme assay The activity of L-triiodothyronine aminotransferase was assayed by the procedure of Soffer and co-workers (1,2) which utilizes 3,5-dinitroL-tyrosine as substrate. The assay measures the rate of formation of the stable colored compound, 3,5-dinitro-p-hydroxyphenylpyruvic acid, spectrophotometrically as an enol-borate complex. The artificial substrate, 3,5-dinitro-L-tyro-
Endo • 1977 Vol 100 • No 4
sine, is used in place of L-triiodothyronine because of the simplicity and accuracy of the spectrophotometric assay, because it is more economical, and because transaminating activities for both compounds have been copurified during the entire procedure in which the enzyme has been isolated and purified (2). 3,5-Dinitro-Ltyrosine has zero activity relative to tyrosine as a substrate for tyrosine aminotransferase (7). The reaction mixtures of 0.9 ml contained 67 mM Tris-HCl (pH 7.8), 20 mM potassium aketoglutarate, 10 mM 3,5-dinitro-L-tyrosine, 10 mM 2-mercaptoethanol, 20 /iM pyridoxal phosphate, and aliquots of tissue homogenates. Generally, 2 fxl volumes of liver homogenates were used; larger aliquots were required for tissue samples with lower enzyme activity. The reaction mixtures were incubated for 60 min at 37 C, and the reactions stopped by the addition of 1.2 ml of 10% trichloroacetic acid. The precipitates were removed by centrifugation at 2000 x g for 10 min. Aliquots of 1.5 ml were removed from each supernatant solution and mixed with an equal volume of a solution containing 2M sodium arsenate and 1M sodium borate, pH 6.5. All tubes were incubated for 30 min at room temperature, and the concentration of the enol-borate complex of 3,5-dinitro-phydroxyphenylpyruvic acid was determined spectrophotometrically at 320 nm, using a molar extinction coefficient of 12,750 (2). Blank samples containing no enzyme were prepared and assayed similarly. One unit of enzyme activity is defined as that amount which results in the formation of 1 /xmol of 3,5-dinitro-p-hydroxyphenylpyruvic acid per min under standard assay conditions. In the assay the molar amounts of glutamic acid and 3,5dinitro-p-hydroxyphenylpyruvic acid formed are equivalent (2). Determination of protein was done by the method of Lowry et at. (8), in which a solution of bovine serum albumin was used as a standard. Each experiment was carried out in its entirety three or more times. Direct effects of thyroid hormones in vitro have not been observed other than as substrates for the enzyme.
Results
Distribution of L-triiodothyronine aminotransferase activity In order to determine the distribution of enzyme activity in the rat, measurements
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 02:17 For personal use only. No other uses without permission. . All rights reserved.
1057
HEPATIC T3 AMINOTRANSFERASE ACTIVITY
TABLE 1. Distribution of L-triiodothyronine aminotransferase activity in various rat organs; results are shown in samples pooled from 2 male rats aged 90 days
were made in various organs and tissues pooled from 2 male rats aged 90 days. As is shown in Table 1, the highest enzyme activity per mg protein was demonstrable in kidney, followed by liver. Enzyme activity in whole brain was less than 10% of that in kidney and liver. Lung, heart, testes and spleen had low but measurable enzyme activity. Virtually no activity was demonstrable in skeletal muscle, thyroid gland, pituitary gland or blood serum.
Hepatic L-triiodothyronine aminotransferase activity in relation to age Measurements were made of hepatic Ltriiodothyronine aminotransferase activity in rats ranging in age from several hours after birth to 290 days. As shown in Fig. 1, enzyme activity remains fairly stable for the first few weeks afterbirth. Thereafter, progressive increases in enzyme activity occur until an apparent peak is reached at 80-120 days of age. In rats older than 120 days, enzyme activity begins to decrease, and levels recorded at 290 days were not significantly greater than at birth.
Organ
L-triiodothyronine aminotransferase activity (milliunits/mg protein)
Kidney Liver Brain Heart Lung Testes Spleen Other organs*
40.5 17.7 0.121 0.050 0.043 0.031 0.016
