0013-7227/91/1296-2881$03.00/0 Endocrinology Copyright (c) 1991 by The Endocrine Society
Vol. 129, No. 6 Printed in U.S.A.
Prolonged Fasting Reduces Rat Hepatic #1 Thyroid Hormone Receptor Protein without Changing the Level of Its Messenger Ribonucleic Acid* JAMES T. LANE, MADAN GODBOLEf, KEVIN A. STRAIT, HAROLD L. SCHWARTZ, AND JACK H. OPPENHEIMER Section of Endocrinology and Metabolism, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
ABSTRACT. The level of hepatic nuclear T3-binding capacity falls in rats subjected to fasting. To define the mechanism underlying these changes, we have assayed in liver the concentration of the mRNA coding for the /31 -receptor (/31-TR) isoform, the total nuclear T3-binding capacity, and the fraction of the total binding capacity that can be specifically immunoprecipitated with an anti-/31-TR immunoglobulin G preparation. Although no changes in /31-TR mRNA concentration were noted,
S
TUDIES from several laboratories have shown that in the rat three mRNAs code for T 3 receptors capable of binding T 3 and mediating T 3 regulation in gene transfection experiments (1-6). These have been designated al-TR, ,31-TR, and 02-TR. The mRNAs for both al-TR and 01-TR are widely distributed among rat tissues, but in liver, heart, and kidney, the j81 mRNA is predominant (7). Alternate splicing of the a-TR gene transcript is also responsible for the generation of two closely related mRNAs, collectively designated a2-TR, which code for a protein lacking a T3-binding domain but capable of interfering with the function of a cotransfected T 3 receptor (8-10). We have recently developed methods that allow us both to estimate the tissue content of specific mRNAs (7) and by immunoprecipitation with a specific anti-01 immunoglobulin G (IgG) to quantitate the level of the ]81-TR (11). The availability of these techniques has prompted us to define the relationship between the hepatic jSl-TR mRNA and protein under various path'oReceived June 20,1991. Address all correspondence and requests for reprints to: Dr. Jack H. Oppenheimer, Division of Endocrinology and Metabolism, Department of Medicine, University of Minnesota, Box 91 Mayo, 515 Delaware Street SE, Minneapolis, Minnesota 55455. * This work was supported by NIH Grant DK-19812 (to J.H.O.), National Research Scientist Award DK-08066 (to K.A.S.), and National Research Scientist Award DK-08427 (to J.T.L.). t Supported by a Boyscast Fellowship (SP/BY/024/87) from the Department of Science and Technology, Government of India.
we observed a 60% fall in total binding capacity. /31-TR mRNA levels were preserved despite a 50% fall in total poly(A)+ RNA. The fall in /31-TR protein, however, was consistent with a generalized decrease in total hepatic protein content. This study provides yet another instance in which measurement of receptor mRNA is not consonant with the behavior of the nuclear T3 receptor protein. {Endocrinology 129: 2881-2885, 1991)
physiological settings. In particular, we were interested in investigating the mechanism underlying the decrease in hepatic T 3 nuclear binding capacity after prolonged fasting, a widely used stimulus for modulating hepatic receptor number (12-16). Reduction in T3-binding capacity may have adaptive value to the fasting animal and synergize with the concomitant reduction in circulating T 3 in attenuating certain T3-regulated metabolic processes (17). Examination of the mRNA-receptor relationships appeared to be particularly appropriate in liver, since the preponderance of TR mRNA and receptor appeared to consist of the /31 isoform, for which a specific antiserum had been developed (11). Materials and Methods Male Sprague-Dawley rats, weighing 200-225 g, were purchased from Bio-Lab (White Bear Lake, MN). Groups of rats were fed Purina chow (Ralston-Purina, St. Louis, MO) ad libitum or were fasted for 5 days. Both groups of animals were allowed unlimited access to water. Animals were killed by exsanguination under ether anesthesia. Livers were quickly removed and immediately placed in liquid nitrogen. The frozen livers were stored at -80 C until further use. The protocol for these studies was reviewed and approved by the University Animal Care Committee of the University of Minnesota. Total RNA extraction was accomplished with guanidine hydrochloride by the method of Chirgwin et ai (18). Northern analyses were performed for all samples, using 20 /ug total RNA (19). To assay for /31-TR mRNA, blots were hybridized with a
2881
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EFFECT OF FASTING ON HEPATIC /tt-TR
2882 32
P-labeled 2.3-kilobase EcoRl cDNA obtained from Murray et al. (4), and to assay for al-TR mRNA, blots were hybridized with the cDNA probe of Thompson et al. (3). Blots were autoradiographed on Kodak XAR-5 film (Eastman Kodak, Rochester, NY) and quantitated by computer-assisted videodensitometry (20). In addition, the blots were hybridized with the cDNA probe for a-actin, kindly provided by Dr. M. Getz (Mayo Clinic, Rochester, MN). The absolute content of the specific TR mRNAs was measured by methods previously described (7). Briefly, the specific mRNA content of a preparation of total brain RNA was predetermined by RNAse protection assay. An aliquot of this preparation was applied to each Northern gel analysis as an internal reference. From the relative optical densities of the test mRNA sample and that of the reference sample, the known mRNA content of the standard, and the RNA/DNA ratio of the tissue, the femtomoles of specific mRNA per mg DNA were calculated. The total poly(A)+ RNA was assayed using an Si nuclease protection assay (21). Pure poly (A) (Boehringer, Mannheim, Germany) was used to construct a standard curve. Total poly(A)+ RNA was calculated on the assumption that poly(A) accounts, on the average, for 7.5% of the mass of hepatic poly(A)+ RNA (21). To determine the hepatic total RNA/DNA ratio, 1 g tissue was homogenized with 19 vol ice-cold water in a glass-Teflon homogenizer. A 5.0-ml aliquot was analyzed for RNA content by the method of Fleck and Munro (22). A 1.0-ml aliquot was analyzed for DNA content by the method of Giles and Meyer (23). Total protein was measured in liver homogenate by the method of Lowry et al. (24) and expressed per mg DNA. The hepatic nuclear T3-binding capacity was determined in isolated nuclei by methods previously described (25). To determine the fraction of the binding capacity accounted for by /31TR protein, T3-binding proteins were extracted from isolated liver nuclei (26), and 0.45 ml of each nuclear extract was incubated at 4 C overnight with 100 ng of specific anti-/31-TR IgG, IgG prepared from preimmune serum, or buffer (25 mM Tris, pH 8.0). The preparation and characterization of the anti/31-TR antiserum have been previously described (11). Immune complexes were cleared by incubation with BSA-blocked protein-A {S. aureus cells, Boehringer Mannheim) for 20 min on ice and centrifuged for 15 min at 5000 X g, and the supernatant was removed. The binding capacity remaining in the supernatants was determined by saturation analysis (25). The data were analyzed by analysis of variance, with P < 0.05 considered significant. Where appropriate, results of experiments were expressed as the mean ± SD. Lines on Scatchard plots were generated by the method of least squares.
Results As noted in previous studies (12-16), fasting resulted in the anticipated decrease in nuclear binding capacity. A representative experiment, showing a 50% fall in binding capacity, is illustrated in Fig. 1. The mean reduction for the entire series was 60%, from 0.89 ± 0.15 to 0. 35 ± 0.08 pmol/mg DNA (n = 14/group). To determine
Endo«1991 Voll29«No6
0.4
0.3.
Fed D Fasted • T3 C 3 O CO 0.1
0.0 0.0
0.2
0.4
0.6
0.8
1.0
Bound (pmol/mg DNA) FIG. 1. Effect of fasting on hepatic nuclear T3-binding capacity. Animals were fasted for 5 days or allowed Purina chow ad libitum. The binding capacity was measured using isolated nuclei (25). Each point represents the mean of duplicates, which varied by less than 10%. FASTED
FED
28 S
18 S
FIG. 2. Effect of fasting on hepatic content of (Sl-TR mRNA. Shown is an autoradiograph of a Northern blot analysis of total RNA (20 jig/ lane) from livers of fed (lanes 1-4) and fasted (lanes 5-8) rats. Blots were hybridized with the 2.3-kilobase cDNA probe for rat /31-TR (4).
whether this decrease in receptor concentration could be attributed to a fall in the level of the mRNAs coding for the receptors, Northern gel analysis was performed using a constant 20 /xg total RNA/sample. In the initial experiment, illustrated in Fig. 2, the intensity of the /31-TR mRNA surprisingly appeared to be almost twice as strong in samples from fasted compared to fed animals. To confirm these findings, studies were performed with 3 additional groups of animals for a total of 16 fed and 17 fasted animals. The combined results of all studies are presented in Table 1. The optical density per 20 ixg RNA of the 01-TR band rose by almost 2-fold from 1.56 ± 0.53
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EFFECT OF FASTING ON HEPATIC 01-TR TABLE 1. 01-TR mRNA levels in rat liver Treatment n Fed Fasted
RNA/DNA 01 mRNA 01 mRNA (fmol/mgRNA) (mg/mg DNA) (fmol/mg DNA)
0.82 ± 0.28 3.24 ± 0.61 2.67 ± 1.06 1.52 ± 0.37* 2.31 ± 1.33 1.53 ± 0.77° Quantitation of 01-TR mRNA concentration in livers of fed and fasted rats. Calculation of the absolute concentration of 01-TR mRNA was carried out as previously described (7). Values are the mean ± SD. °P
Vol. 129, No. 6 Printed in U.S.A.
Prolonged Fasting Reduces Rat Hepatic #1 Thyroid Hormone Receptor Protein without Changing the Level of Its Messenger Ribonucleic Acid* JAMES T. LANE, MADAN GODBOLEf, KEVIN A. STRAIT, HAROLD L. SCHWARTZ, AND JACK H. OPPENHEIMER Section of Endocrinology and Metabolism, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
ABSTRACT. The level of hepatic nuclear T3-binding capacity falls in rats subjected to fasting. To define the mechanism underlying these changes, we have assayed in liver the concentration of the mRNA coding for the /31 -receptor (/31-TR) isoform, the total nuclear T3-binding capacity, and the fraction of the total binding capacity that can be specifically immunoprecipitated with an anti-/31-TR immunoglobulin G preparation. Although no changes in /31-TR mRNA concentration were noted,
S
TUDIES from several laboratories have shown that in the rat three mRNAs code for T 3 receptors capable of binding T 3 and mediating T 3 regulation in gene transfection experiments (1-6). These have been designated al-TR, ,31-TR, and 02-TR. The mRNAs for both al-TR and 01-TR are widely distributed among rat tissues, but in liver, heart, and kidney, the j81 mRNA is predominant (7). Alternate splicing of the a-TR gene transcript is also responsible for the generation of two closely related mRNAs, collectively designated a2-TR, which code for a protein lacking a T3-binding domain but capable of interfering with the function of a cotransfected T 3 receptor (8-10). We have recently developed methods that allow us both to estimate the tissue content of specific mRNAs (7) and by immunoprecipitation with a specific anti-01 immunoglobulin G (IgG) to quantitate the level of the ]81-TR (11). The availability of these techniques has prompted us to define the relationship between the hepatic jSl-TR mRNA and protein under various path'oReceived June 20,1991. Address all correspondence and requests for reprints to: Dr. Jack H. Oppenheimer, Division of Endocrinology and Metabolism, Department of Medicine, University of Minnesota, Box 91 Mayo, 515 Delaware Street SE, Minneapolis, Minnesota 55455. * This work was supported by NIH Grant DK-19812 (to J.H.O.), National Research Scientist Award DK-08066 (to K.A.S.), and National Research Scientist Award DK-08427 (to J.T.L.). t Supported by a Boyscast Fellowship (SP/BY/024/87) from the Department of Science and Technology, Government of India.
we observed a 60% fall in total binding capacity. /31-TR mRNA levels were preserved despite a 50% fall in total poly(A)+ RNA. The fall in /31-TR protein, however, was consistent with a generalized decrease in total hepatic protein content. This study provides yet another instance in which measurement of receptor mRNA is not consonant with the behavior of the nuclear T3 receptor protein. {Endocrinology 129: 2881-2885, 1991)
physiological settings. In particular, we were interested in investigating the mechanism underlying the decrease in hepatic T 3 nuclear binding capacity after prolonged fasting, a widely used stimulus for modulating hepatic receptor number (12-16). Reduction in T3-binding capacity may have adaptive value to the fasting animal and synergize with the concomitant reduction in circulating T 3 in attenuating certain T3-regulated metabolic processes (17). Examination of the mRNA-receptor relationships appeared to be particularly appropriate in liver, since the preponderance of TR mRNA and receptor appeared to consist of the /31 isoform, for which a specific antiserum had been developed (11). Materials and Methods Male Sprague-Dawley rats, weighing 200-225 g, were purchased from Bio-Lab (White Bear Lake, MN). Groups of rats were fed Purina chow (Ralston-Purina, St. Louis, MO) ad libitum or were fasted for 5 days. Both groups of animals were allowed unlimited access to water. Animals were killed by exsanguination under ether anesthesia. Livers were quickly removed and immediately placed in liquid nitrogen. The frozen livers were stored at -80 C until further use. The protocol for these studies was reviewed and approved by the University Animal Care Committee of the University of Minnesota. Total RNA extraction was accomplished with guanidine hydrochloride by the method of Chirgwin et ai (18). Northern analyses were performed for all samples, using 20 /ug total RNA (19). To assay for /31-TR mRNA, blots were hybridized with a
2881
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EFFECT OF FASTING ON HEPATIC /tt-TR
2882 32
P-labeled 2.3-kilobase EcoRl cDNA obtained from Murray et al. (4), and to assay for al-TR mRNA, blots were hybridized with the cDNA probe of Thompson et al. (3). Blots were autoradiographed on Kodak XAR-5 film (Eastman Kodak, Rochester, NY) and quantitated by computer-assisted videodensitometry (20). In addition, the blots were hybridized with the cDNA probe for a-actin, kindly provided by Dr. M. Getz (Mayo Clinic, Rochester, MN). The absolute content of the specific TR mRNAs was measured by methods previously described (7). Briefly, the specific mRNA content of a preparation of total brain RNA was predetermined by RNAse protection assay. An aliquot of this preparation was applied to each Northern gel analysis as an internal reference. From the relative optical densities of the test mRNA sample and that of the reference sample, the known mRNA content of the standard, and the RNA/DNA ratio of the tissue, the femtomoles of specific mRNA per mg DNA were calculated. The total poly(A)+ RNA was assayed using an Si nuclease protection assay (21). Pure poly (A) (Boehringer, Mannheim, Germany) was used to construct a standard curve. Total poly(A)+ RNA was calculated on the assumption that poly(A) accounts, on the average, for 7.5% of the mass of hepatic poly(A)+ RNA (21). To determine the hepatic total RNA/DNA ratio, 1 g tissue was homogenized with 19 vol ice-cold water in a glass-Teflon homogenizer. A 5.0-ml aliquot was analyzed for RNA content by the method of Fleck and Munro (22). A 1.0-ml aliquot was analyzed for DNA content by the method of Giles and Meyer (23). Total protein was measured in liver homogenate by the method of Lowry et al. (24) and expressed per mg DNA. The hepatic nuclear T3-binding capacity was determined in isolated nuclei by methods previously described (25). To determine the fraction of the binding capacity accounted for by /31TR protein, T3-binding proteins were extracted from isolated liver nuclei (26), and 0.45 ml of each nuclear extract was incubated at 4 C overnight with 100 ng of specific anti-/31-TR IgG, IgG prepared from preimmune serum, or buffer (25 mM Tris, pH 8.0). The preparation and characterization of the anti/31-TR antiserum have been previously described (11). Immune complexes were cleared by incubation with BSA-blocked protein-A {S. aureus cells, Boehringer Mannheim) for 20 min on ice and centrifuged for 15 min at 5000 X g, and the supernatant was removed. The binding capacity remaining in the supernatants was determined by saturation analysis (25). The data were analyzed by analysis of variance, with P < 0.05 considered significant. Where appropriate, results of experiments were expressed as the mean ± SD. Lines on Scatchard plots were generated by the method of least squares.
Results As noted in previous studies (12-16), fasting resulted in the anticipated decrease in nuclear binding capacity. A representative experiment, showing a 50% fall in binding capacity, is illustrated in Fig. 1. The mean reduction for the entire series was 60%, from 0.89 ± 0.15 to 0. 35 ± 0.08 pmol/mg DNA (n = 14/group). To determine
Endo«1991 Voll29«No6
0.4
0.3.
Fed D Fasted • T3 C 3 O CO 0.1
0.0 0.0
0.2
0.4
0.6
0.8
1.0
Bound (pmol/mg DNA) FIG. 1. Effect of fasting on hepatic nuclear T3-binding capacity. Animals were fasted for 5 days or allowed Purina chow ad libitum. The binding capacity was measured using isolated nuclei (25). Each point represents the mean of duplicates, which varied by less than 10%. FASTED
FED
28 S
18 S
FIG. 2. Effect of fasting on hepatic content of (Sl-TR mRNA. Shown is an autoradiograph of a Northern blot analysis of total RNA (20 jig/ lane) from livers of fed (lanes 1-4) and fasted (lanes 5-8) rats. Blots were hybridized with the 2.3-kilobase cDNA probe for rat /31-TR (4).
whether this decrease in receptor concentration could be attributed to a fall in the level of the mRNAs coding for the receptors, Northern gel analysis was performed using a constant 20 /xg total RNA/sample. In the initial experiment, illustrated in Fig. 2, the intensity of the /31-TR mRNA surprisingly appeared to be almost twice as strong in samples from fasted compared to fed animals. To confirm these findings, studies were performed with 3 additional groups of animals for a total of 16 fed and 17 fasted animals. The combined results of all studies are presented in Table 1. The optical density per 20 ixg RNA of the 01-TR band rose by almost 2-fold from 1.56 ± 0.53
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EFFECT OF FASTING ON HEPATIC 01-TR TABLE 1. 01-TR mRNA levels in rat liver Treatment n Fed Fasted
RNA/DNA 01 mRNA 01 mRNA (fmol/mgRNA) (mg/mg DNA) (fmol/mg DNA)
0.82 ± 0.28 3.24 ± 0.61 2.67 ± 1.06 1.52 ± 0.37* 2.31 ± 1.33 1.53 ± 0.77° Quantitation of 01-TR mRNA concentration in livers of fed and fasted rats. Calculation of the absolute concentration of 01-TR mRNA was carried out as previously described (7). Values are the mean ± SD. °P
