Triiodothyronine Receptors During Maturation LESLIE J. D E G R O O T , MARILYN ROBERTSON, AND PAUL A. RUE Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Illinois of T3-receptor complex, mitochondrial a-glycerophosphate dehydrogenase activity changes relatively less during maturation. Administration of estrogen, testosterone, and dexamethasone to immature female rats did not alter NTBP capacity or affinity. (Endocrinology 100: 1511, 1977)
ABSTRACT. Capacity of rat liver nuclear triiodothyronine (T3) binding protein (NTBP) for T3 more than quadruples from birth through 50-120 days, whether related to DNA or weight of tissue. Ka for T3 doubles during this interval. T3 content of nuclei nearly parallels capacity. The proportion of receptors occupied by T3 is 0.3 at birth, falling to .16 in old rats. In contrast to these changes in liver content
V
ERY little is known about the development or physiology of the putative thyroid hormone receptors found in rat liver nuclei. It is reported that serum T4 and T3 are low in the neonatal rat, climb to adult levels over 30 days (1,2), and that serum T4 declines in aging males (2). Liver mitochondrial alphaglycerophosphate dehydrogenase (a-GPDH) activity is reported to be lower in neonates than in normal adult animals, but is responsive to T3 stimulation, even in embryonic or neonatal animals (3). The following studies were undertaken to characterize the development of nuclear receptors for T3 during maturation, the effect of certain hormones on receptor capacity, and possible correlations of receptor function with physiologic responses to thyroid hormone. We find that nuclear receptor capacity is low at birth and maximal at 50-80 days. These changes parallel T3 content of liver, but not a-GPDH activity or serum T3 concentrations. Materials and Methods Experiments were conducted on Charles River Farm strain rats of the indicated ages and sexes. For each point in each comparison, pooled livers of 4-16 neonatal animals were used, as required to provide sufficient tissue, and pools of 2-3 adult rat livers were employed. Received April 19, 1976. Supported in part by United States Public Health Service Grants AM-13,377 and CA-14,599.
Liver T3 binding affinity and capacity were measured by incubation of nuclei with [125I]T3, and increasing amounts of unlabelled T3, as described (4). Data for each Scatchard analysis included T3 binding at seven concentrations, and saturating levels of T3, each assayed in triplicate. a-GPDH activity of mitochondria was measured as described by Lee and Lardy (5). T3 content of serum, liver homogenate, and nuclei was measured by RIA. Aliquots of homogenate and nuclei were extracted with ethanol, extracts were dried, and the extracted T3 was taken up in 1% bovine serum albumin + . 1 4 M NaCl (6). Recovery of T3, assessed by extraction of [125I]T3 administered in vivo, was 80-95%. The RIA for T3 employed a double antibody technique and 8anilino-1-naphthalene sulfonic acid as a displacing agent. Serum samples were run against blanks and standards containing an equivalent amount of T3-free serum (rat and human sera gave equivalent results). Tissue extracts were run against blanks and standards containing equivalent albumin-NaCl solution. Administration of hormones is described below. DNA was determined by the method of Munro and Fleck (7), and protein by the method of Lowry et al. (8). Statistical comparisons were made by a twotailed Student t test using an Olivetti P-602 calculator programmed for two unrelated samples.
Results
T3 binding capacity and affinity of rat liver nuclei T3 binding capacity was low at birth and increased progressively during the postnatal month (Table 1). From 50 to 120 days
1511
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:42 For personal use only. No other uses without permission. . All rights reserved.
1512
D E G R O O T , ROBERTSON AND RUE
I 06
ill a
2
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o
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+1
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2 8
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5
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Endo • 1977 Vol 100 . No 6'
capacity was 42-48 pg/100 /u,g DNA, more than four-fold the neonatal level. Compared to animals of 50-120 days, older males had on the average a lower capacity, but the difference did not reach statistical significance. Similar observations were found when the data was expressed as T3 binding capacity per gram liver (Table 1). The affinity constant for T3 binding to nuclear receptors in adult males was .18 - .25 x 1010M-\ and is similar to that reported previously (0.2 x lO^M"1) (4,9). Ka for T3 was significantly lower in immature animals (0-13 days) than in animals of 50-80 days. Various technical problems that might influence these results must be considered. The liver of neonatal rats is very fragile and nuclei are easily fragmented. To avoid damage, this tissue was homogenized oneeighth as long as adult tissue, and handled especially gently in each step of processing. Final preparations showed well preserved nuclei under phase microscopy that were free of cytoplasmic tags and debris. The weight of newborn liver used ranged from 1.4 to 5 g during the first 14 days, when livers of pooled litter mates were utilized. The fig DNA used per tube in the T3 binding experiments was equivalent to that used from the adult animals. Samples for DNA analysis were obtained directly from the final nuclear suspension used in the experimental procedures for T3 binding. Finally, it has been shown in our laboratory that NTBP leaks from nuclei under the conditions of incubation employed in this and other laboratories (10). Variations in leakage of NTBP to medium could alter the determination of capacity. This NTBP can be quantitatively recovered in the incubation medium, and its capacity for binding T3 can be estimated at the end of incubation, by use of an anion exchange resin test (11). To compare NTBP leakage during incubation, we measured fractional binding of T3 to NTBP in the supernatant after in vitro incubation of nuclei at one level of T3 in each, experimental study. The bound fraction in medium from infant and adult prep-
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T, RECEPTORS DURING MATURATION arations was nearly identical in a series of ten experiments, averaging 18% in tubes containing 3 nM T3. Thus, there was no excess leak of receptor from the infant rat nuclei to the incubation medium, in comparison to adult rat nuclei. Variable loss of nuclear T 3 receptors to the soluble fraction of the homogenate during processing is also a theoretical possibility and has not been specifically disproven in these experiments. In fact, it would be essentially impossible to test this hypothesis directly. However, it is known that nuclear T3 receptors do not leak from nucleus to incubation fluid during incubation in vitro at 0 C in buffer similar to that used in homogenization and differential centrifugation (12). Thus, we believe this problem can be discounted. T3 content of liver
Endogenous [127I]T3 was evaluated in rat serum, and in ethanol extracts of liver homogenate and nuclei. Serum T 3 was .76-.78 ng/ml in 20-210 day male rats. Nuclear and homogenate T3 concentration increased 3-4-fold from 0 - 6 days to 50-120 days, and decreased in older rats (Table 1). "Apparent saturation" of receptors It is of interest to estimate saturation of receptors in the different age groups. Content of T 3 was directly measured on ethanol extracts of nuclei. Total content of T^g liver was corrected for average recovery of 60% of nuclei in the preparations. Binding capacity was measured directly, and also corrected for recovery of 60% of nuclei (12). Estimated binding capacity was also corrected for the average 50% loss of NTBP to medium occurring during the incubation period (10). In 50 to 120 day old animals total estimated capacity is thus 2.2-2.4 ng T^g tissue. These data are simiar to our previous estimates of binding capacity determined from in vitro studies, and the 2.4 ng/g capacity determined on adult male animals by in vivo studies (12).
1513
12 w 10
ee o. < o o Q
z CD
NUCLEAR CONTENT OF T 3 /g LIVER (pgxld 2 ) FIG. 1. Relation of nuclear binding capacity for T3 to nuclear content of T3, including data from all age groups.
Thus the "apparent saturation" determined from these data obtained in vitro probably reasonably estimates the conditions existing in vivo. Further, comparisons between age groups studied in the same manner should be valid. The "apparent saturation" was .3 in neonatal animals, and .22-. 16 in mature animals. There is a striking correlation between T3 content and capacity for binding T3 (Fig. 1). a-GPDH activity (Table 2) Mitochondria were prepared from animals, and assayed for a-GPDH as described (5). These experiments were conducted on the same animals used in the study of nuclear T 3 binding or T3 content. Original data are expressed as O.D. change/min/mg protein. Some assays were conducted on groups of mitochondrial samples frozen after preparation and before determination of a-GPDH, and these results were uniformly lower than those obtained in fresh tissue preparations. However, the relationship of activity in immature vs. mature animals was similar. To allow comparison of groups of animals studied on many different occasions, and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:42 For personal use only. No other uses without permission. . All rights reserved.
1514
TABLE 2. aGPDH activity at various ages
Group
Age (Days)
N
1 o
0-6
5 3 4 8 3 6 4 4 4
3 4 5
6 7 S 9
End.) 1977 Vol 100 . No 6
D E G R O O T , ROBERTSON AND RUE
9-13 14-22 28-40 50-60 70-90 90- 150 150+ Tliyroklectomized
aGPDH AO.D./inin/mg protein, as fraction ol Croup 4
P r.v. Croup 4
0.77 ± 0.24 0.63 ± 0.06 1.33 ± 0.27
ABSTRACT. Capacity of rat liver nuclear triiodothyronine (T3) binding protein (NTBP) for T3 more than quadruples from birth through 50-120 days, whether related to DNA or weight of tissue. Ka for T3 doubles during this interval. T3 content of nuclei nearly parallels capacity. The proportion of receptors occupied by T3 is 0.3 at birth, falling to .16 in old rats. In contrast to these changes in liver content
V
ERY little is known about the development or physiology of the putative thyroid hormone receptors found in rat liver nuclei. It is reported that serum T4 and T3 are low in the neonatal rat, climb to adult levels over 30 days (1,2), and that serum T4 declines in aging males (2). Liver mitochondrial alphaglycerophosphate dehydrogenase (a-GPDH) activity is reported to be lower in neonates than in normal adult animals, but is responsive to T3 stimulation, even in embryonic or neonatal animals (3). The following studies were undertaken to characterize the development of nuclear receptors for T3 during maturation, the effect of certain hormones on receptor capacity, and possible correlations of receptor function with physiologic responses to thyroid hormone. We find that nuclear receptor capacity is low at birth and maximal at 50-80 days. These changes parallel T3 content of liver, but not a-GPDH activity or serum T3 concentrations. Materials and Methods Experiments were conducted on Charles River Farm strain rats of the indicated ages and sexes. For each point in each comparison, pooled livers of 4-16 neonatal animals were used, as required to provide sufficient tissue, and pools of 2-3 adult rat livers were employed. Received April 19, 1976. Supported in part by United States Public Health Service Grants AM-13,377 and CA-14,599.
Liver T3 binding affinity and capacity were measured by incubation of nuclei with [125I]T3, and increasing amounts of unlabelled T3, as described (4). Data for each Scatchard analysis included T3 binding at seven concentrations, and saturating levels of T3, each assayed in triplicate. a-GPDH activity of mitochondria was measured as described by Lee and Lardy (5). T3 content of serum, liver homogenate, and nuclei was measured by RIA. Aliquots of homogenate and nuclei were extracted with ethanol, extracts were dried, and the extracted T3 was taken up in 1% bovine serum albumin + . 1 4 M NaCl (6). Recovery of T3, assessed by extraction of [125I]T3 administered in vivo, was 80-95%. The RIA for T3 employed a double antibody technique and 8anilino-1-naphthalene sulfonic acid as a displacing agent. Serum samples were run against blanks and standards containing an equivalent amount of T3-free serum (rat and human sera gave equivalent results). Tissue extracts were run against blanks and standards containing equivalent albumin-NaCl solution. Administration of hormones is described below. DNA was determined by the method of Munro and Fleck (7), and protein by the method of Lowry et al. (8). Statistical comparisons were made by a twotailed Student t test using an Olivetti P-602 calculator programmed for two unrelated samples.
Results
T3 binding capacity and affinity of rat liver nuclei T3 binding capacity was low at birth and increased progressively during the postnatal month (Table 1). From 50 to 120 days
1511
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:42 For personal use only. No other uses without permission. . All rights reserved.
1512
D E G R O O T , ROBERTSON AND RUE
I 06
ill a
2
"Sb
•5b
o
—
+1 ~
in
to
+1
+1
+1
+1
2 8
.5 Q — Q
H 2
3
5
+1 +1 oo oo
a; - a a c— X
C8
3
OQ
Endo • 1977 Vol 100 . No 6'
capacity was 42-48 pg/100 /u,g DNA, more than four-fold the neonatal level. Compared to animals of 50-120 days, older males had on the average a lower capacity, but the difference did not reach statistical significance. Similar observations were found when the data was expressed as T3 binding capacity per gram liver (Table 1). The affinity constant for T3 binding to nuclear receptors in adult males was .18 - .25 x 1010M-\ and is similar to that reported previously (0.2 x lO^M"1) (4,9). Ka for T3 was significantly lower in immature animals (0-13 days) than in animals of 50-80 days. Various technical problems that might influence these results must be considered. The liver of neonatal rats is very fragile and nuclei are easily fragmented. To avoid damage, this tissue was homogenized oneeighth as long as adult tissue, and handled especially gently in each step of processing. Final preparations showed well preserved nuclei under phase microscopy that were free of cytoplasmic tags and debris. The weight of newborn liver used ranged from 1.4 to 5 g during the first 14 days, when livers of pooled litter mates were utilized. The fig DNA used per tube in the T3 binding experiments was equivalent to that used from the adult animals. Samples for DNA analysis were obtained directly from the final nuclear suspension used in the experimental procedures for T3 binding. Finally, it has been shown in our laboratory that NTBP leaks from nuclei under the conditions of incubation employed in this and other laboratories (10). Variations in leakage of NTBP to medium could alter the determination of capacity. This NTBP can be quantitatively recovered in the incubation medium, and its capacity for binding T3 can be estimated at the end of incubation, by use of an anion exchange resin test (11). To compare NTBP leakage during incubation, we measured fractional binding of T3 to NTBP in the supernatant after in vitro incubation of nuclei at one level of T3 in each, experimental study. The bound fraction in medium from infant and adult prep-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:42 For personal use only. No other uses without permission. . All rights reserved.
T, RECEPTORS DURING MATURATION arations was nearly identical in a series of ten experiments, averaging 18% in tubes containing 3 nM T3. Thus, there was no excess leak of receptor from the infant rat nuclei to the incubation medium, in comparison to adult rat nuclei. Variable loss of nuclear T 3 receptors to the soluble fraction of the homogenate during processing is also a theoretical possibility and has not been specifically disproven in these experiments. In fact, it would be essentially impossible to test this hypothesis directly. However, it is known that nuclear T3 receptors do not leak from nucleus to incubation fluid during incubation in vitro at 0 C in buffer similar to that used in homogenization and differential centrifugation (12). Thus, we believe this problem can be discounted. T3 content of liver
Endogenous [127I]T3 was evaluated in rat serum, and in ethanol extracts of liver homogenate and nuclei. Serum T 3 was .76-.78 ng/ml in 20-210 day male rats. Nuclear and homogenate T3 concentration increased 3-4-fold from 0 - 6 days to 50-120 days, and decreased in older rats (Table 1). "Apparent saturation" of receptors It is of interest to estimate saturation of receptors in the different age groups. Content of T 3 was directly measured on ethanol extracts of nuclei. Total content of T^g liver was corrected for average recovery of 60% of nuclei in the preparations. Binding capacity was measured directly, and also corrected for recovery of 60% of nuclei (12). Estimated binding capacity was also corrected for the average 50% loss of NTBP to medium occurring during the incubation period (10). In 50 to 120 day old animals total estimated capacity is thus 2.2-2.4 ng T^g tissue. These data are simiar to our previous estimates of binding capacity determined from in vitro studies, and the 2.4 ng/g capacity determined on adult male animals by in vivo studies (12).
1513
12 w 10
ee o. < o o Q
z CD
NUCLEAR CONTENT OF T 3 /g LIVER (pgxld 2 ) FIG. 1. Relation of nuclear binding capacity for T3 to nuclear content of T3, including data from all age groups.
Thus the "apparent saturation" determined from these data obtained in vitro probably reasonably estimates the conditions existing in vivo. Further, comparisons between age groups studied in the same manner should be valid. The "apparent saturation" was .3 in neonatal animals, and .22-. 16 in mature animals. There is a striking correlation between T3 content and capacity for binding T3 (Fig. 1). a-GPDH activity (Table 2) Mitochondria were prepared from animals, and assayed for a-GPDH as described (5). These experiments were conducted on the same animals used in the study of nuclear T 3 binding or T3 content. Original data are expressed as O.D. change/min/mg protein. Some assays were conducted on groups of mitochondrial samples frozen after preparation and before determination of a-GPDH, and these results were uniformly lower than those obtained in fresh tissue preparations. However, the relationship of activity in immature vs. mature animals was similar. To allow comparison of groups of animals studied on many different occasions, and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:42 For personal use only. No other uses without permission. . All rights reserved.
1514
TABLE 2. aGPDH activity at various ages
Group
Age (Days)
N
1 o
0-6
5 3 4 8 3 6 4 4 4
3 4 5
6 7 S 9
End.) 1977 Vol 100 . No 6
D E G R O O T , ROBERTSON AND RUE
9-13 14-22 28-40 50-60 70-90 90- 150 150+ Tliyroklectomized
aGPDH AO.D./inin/mg protein, as fraction ol Croup 4
P r.v. Croup 4
0.77 ± 0.24 0.63 ± 0.06 1.33 ± 0.27
