Reprinted from Science, June 28, 1963, Vol. 140, No. 3574, pages 1417-1418 Copyright © 1963 by the American Association for the Advancement of Science Thyroxine: Effects on Amino Acid Incorporation into Protein in vivo Abstract: Treatment of rats with L- thyroxine increases the incorporation in vivo of radioactive amino acids into protein of liver, kidney, and heart, but not of spleen, testis, or brain. The dis- tribution of the effect among the organs is the same as that observed in thyroxine stimulation of oxidative metabolism. Thus, stimulation of protein synthesis seems to be a physiological action of the — thyroid hormone. The effects of thyroid hormones on protein biosynthesis have received con- siderable attention in recent years. In 1952, Du Toit (J) observed that liver ‘slices from thyroidectomized rats showed low rates of amino acid incor- poration into protein which could be raised toward normal by treatment of the animals with L-thyroxine (/). Fur- ther evidence of a reduced rate of pro- tein biosynthesis in hypothyroidism and its return to normal after effective thyroid therapy has been obtained in in- tact man by Crispell, Parson, and Holli- field (2); in one euthyroid subject given triiodothyronine, however, pro- tein synthesis appeared to be de- creased. In 1959, Sokoloff and Kauf- man (3) reported that prior adminis- tration of L-thyroxine to normal ani- mals or its addition to the incubation medium directly in vitro stimulated the rate of amino acid incorporation into protein ‘in cell-free rat-liver ho- mogenates (3); in similar prepara- tions from thyroidectomized rats the rate of amino acid incorporation was low (4). On the basis of these and related findings they suggested that the characteristic action of thyroxine on oxygen consumption might be second- ary to the stimulation of energy-requir- ing processes such as protein biosyn- thesis (3). Subsequent studies by these workers and their associates (4, 5) further characterized the . thyroxine stimulation of protein biosynthesis and localized it to the step in protein syn- thesis involving the transfer of soluble RNA-bound amino acid to microsomal protein. The effect of thyroxine added in vitro has now also been observed in slices of rabbit bone marrow by Necheles (6), in cell-free rat liver homogenates by Kamei, Michel, and Roche (7), and in slices of rat kidney by Hanson, Lindsay, and Barker (8). The effect of thyroxine treatment in vivo has been confirmed in cell-free rat liver preparations from thyroidec- tomized rats by Stein and Gross (9) and most recently by Tata and his associates (J0). In all but one of the studies cited, protein synthesis was assayed in vitro. In the one exception (2), the technique employed (JJ) was indirect and al- lowed inferences concerning the rate of protein synthesis only if certain as- sumptions, which might be hazardous to make in the case of hyperthyroidism, are valid. There has, therefore, been no satisfactory demonstration that the thy- roxine stimulation of protein biosyn- thesis assayed in vitro represents a true physiological action of the hormone. In our study, protein biosynthesis in vivo has been examined directly by mea- surement of the rate of incorporation of radioactive amino acids into tissue proteins in the intact animal. The results indicate that the thyroxine stimulation of amino acid incorporation into pro- tein observed in vitro also occurs in vivo in those organs which respond to thyroxine with increased metabolic rates. Normal male Osborne-Mendel rats weighing between 65 and 115 g were paired for age and weight. One mem- ber of each pair received 100 pg of sodium t-thyroxine dissolved in 0.5 or 2.0 ml, of O0OIN. NaOH by intraperitoneal injection daily for 2 or 3 days; the control animal received equivalent volumes of the solvent alone. Both members of each pair were then treated identically through all subse- quent procedures in accordance with paired experimental design. The incor- poration of amino acid into protein was assayed simultaneously in paired ani- mals on the day after the last injection. The animals were either fasted or al- lowed 20 percent glucose in the drink- ing water ad libitum for at least 18 hours immediately before the experi- ment. Amino acid incorporation into pro- tein was determined by measurement of the specific activity of various tissue proteins after the parenteral administra- tion of L-leucine-1-C’*. In most experi- ments the L-leucine-1-C* (specific ac- tivity, 5.7 mc/mmole) was dissolved in normal saline to a final concentration of 1.25 y»c/ml and infused continuously by way of the tail vein at a constant rate of 0.05 ml/min by means of an infusion pump (Harvard Apparatus Co. model No. 600-910) for 30, 60, or 120 minutes. In a few experiments the L- 800 Wiconrroe THYROXINE 600;- See 2. 8 COUNT/ MINUTE PER MG PROTEIN 3 LIVER KIDNEY HEART SPLEEN TESTIS BRAIN Fig. 1. The incorporation of L-leucine-1- C* into protein in six organs of six paired thyroxine-treated and control rats infused intravenously with labeled amino acid for 120 minutes. The values represent means and standard errors. The asterisk indicates Statistically significant difference (p < 0.05 as determined by method of paired com- parison). leucine-1-C* (specific activity, 6.8 mc/mmole) was administered by a single intraperitoneal injection of 2.5 pc of the amino acid in 1 ml of normal saline, and specific activity of the tissue proteins was determined 40 to 60 min- utes after the injection. At the end of the infusion or the specified time, the animals were decapitated; the various organs were removed as rapidly as pos- sible, blotted free of blood, and frozen in liquid nitrogen. A fraction of each organ was then homogenized in 6 per- cent trichloroacetic acid, and the pre- cipitated protein was purified, plated, and assayed for specific activity (3, 4). The results on the six organs ex- amined simultaneously in six pairs of control and thyroxine-treated rats after 120 minutes of infusion of the radioac- tive amino acid are presented in Fig. 1. Thyroxine treatment significantly in- creased incorporation of L-leucine-1- C” into protein in the liver, kidney, and heart. No -significant effects were ob-. served in the spleen, testis, and brain. Thyroxine also does not stimulate amino acid incorporation into protein in cell- free brain preparations from rats of this age (12). Although there is a difference . between the mean values in the spleen,. this difference is not statistically signifi- cant because of great individual varia- tion in the specific activities of spleen protein. The total spleen weight also varied widely. The variations in both total weight and protein specific activity may reflect differences in agonal con- traction and trapping of blood in the organ before its removal. The thyroxine effects seemed unre- Reprinted with permission by the U. S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service Table. 1. Relationship. of mode of amino acid administration to the effects of L-thyroxine on the incorporation of t-leucine-1-C'* into protein in various organs. The values are the mean of individual percentage differences between paired control and thyroxine-treated rats in numbers of paired experiments indicated in parentheses. Duration of Thyroxine effects in various organs amino acid (%) dose (min) Liver Kidney Heart Spleen Testis Brain Continuous. intravenous infusion 30 28% &1) 3--@ 88 (1) —80 (1) —38 (1) Zo": *(1) 60 7 (14) 37 (5) (7) 3 (4) —3 (4) —7 (4) 120 19* (13) 42+ (6) 277 (11) 49 (6) Ty (6) 20 (6) Total 16* (34) 37+ (12) 21719) 20 (11) 2 C91) 10 (11) Single intraperitoneal injection 374434) 28:7'1(3,) 43 (3) 4 (3) 2+ @) 9. (3) “Ft * lated to the duration of the intusion or the mode of administration of the la- beled amino acid. Although in some cases there were insufficient numbers of experiments for adequate statistical analysis, there is evidence (Table 1) of stimulations of the incorporation of L-leucine-1-C* into protein in_ liver, kidney, and heart after each of the periods of continuolis intravenous in- fusion or the single intraperitoneal in- jection. On the other hand, there appear to be no consistent effects of thyroxine in the spleen, testis, or brain regardless of the duration or mode of administra- tion. The thyroxine stimulations in liver were significant in both the fasted and glucose-fed animals but were more con- sistent in the latter, an observation pre- viously made on the effects of thyroxine on other biosynthetic pathways (/3) and attributed to the rapid depletion of liver glycogen in the fasted, thyrotoxic animal. The results after the single intraperi- toneal injection are at variance with those reported in which thyroxine treat- ment appeared to reduce incorporation of the labeled amino acid (14); how- ever, in those studies the protein specific activity was assayed several hours after the injection of amino acid when the ra- dioactivity of the precursor amino acid pool was negligible and protein break- down rather than synthesis was the major determinant of protein specific activity. Increased incorporation of the radio- active amino acid is not in itself conclu- sive evidence that thyroxine stimulates protein biosynthesis in vivo. Incorpo- ration of the radioactive amino acid is dependent not only on protein synthe- sis but also on the history of the specific activity of the precursor amino acid pool. Measurements of the specific ac- p<0.01 , ; 5 . Be 00S' 'f determined by method of paired comparison. tivity of the free leucine in the same tissues in which specific activity of protein was measured are currently in progress. There are, however, already a number of indications that the thyrox- ine stimulation of L-leucine-1-C™ incor- poration into protein cannot be attrib- uted to an effect on pool specific activity. The specific activity of the free-leucine pool in the ‘tissue is de- pendent on the rate of delivery of the L-leucine-1-C relative to the size of the tissue leucine pool as well as to the rate of metabolic turnover of this pool. Circulatory effects would be most apparent early in the infusion procedure and would tend to disappear as the in- fusion continued and the specific ac- tivity of the tissue free-leucine pool approached equilibrium with that of the infused amino acid. However, in these studies the thyroxine effect appears to be unrelated to the duration of infusion or, if anything, more prominent with the prolonged infusions (Table 1). Also the thyroxine effects appear to be simi- lar by two entirely different modes of administration. Evidence in the litera- ture suggests that in thyrotoxicosis, free amino acid or leucine pools in the tis- sues are unchanged or increased (/5), a change which would tend to diminish the incorporation of the labeled aminc acid into protein. Finally, increased turnover of the amino acid pool, which almost certainly occurs in the hyperthy- roid state, would result in continuous dilution of the administered labeled amino acid by unlabeled amino acid from endogenous sources and would, therefore, also tend to diminish the in- corporation into protein of the labeled species. Thus, our results strongly suggest that thyroxine stimulates protein bio- synthesis in vivo as well as in vitro. Furthermore, the pattern of stimulation of amino acid incorporation in various organs is strikingly similar to the pat- tern of effects of thyroxine on oxygen consumption (/6). Thyroxine appears to stimulate both amino acid incorpora- tion and oxygen consumption in the liver, kidney, and heart but has no effect on either process in the spleen, testis, and brain (1/6). The effects on amino acid incorporation, therefore, parallel the effects on an accepted physiological action of the hormone, indicating that the action of thyroxine on protein bio- synthesis, previously observed in vitro is not simply a laboratory curiosity but a true physiological action of the thyroid hormone. Furthermore, it has recently been shown that puromycin inhibition of protein synthesis (and, therefore, also of any thyroxine effect on protein synthesis) acutely reduces the meta- bolic rate in thyrotoxicosis to normal levels (77). This finding suggests that the effects of thyroxine on oxidative metabolism are secondary to its effects on protein synthesis. RoBERT MICHELS JOHN CASON Louis SOKOLOFF Laboratory of Clinical Science, National Institute of Mental Health, Bethesda 14, Maryland References 1. C. H. DuToit. in A Symposium on Phos- phorus Metabolism, W. D. McElroy and B. Glass, Eds. (Johns Hopkins Univ. Press, Baltimore, 1952), vol. 2, p. 597. 2. K. R. Crispell, W: Parson, G. Hollifield, J. Clin, Invest. 35, 164 (1956). 3. L. Sokoloff and S. Kaufman, 569 (1959). , J. Biol. Chem. 236, 795 (1961). . L. Sokoloff, S. Kaufman,. H. V. Gelboin, Biochim. Biophys. Acta 52, 410 (1961); L. Sokoloff, S. Kaufman, P. L. Campbell, C. M. Francis, H. V. Gelboin, J. Biol. Chem. 238, 1432 (1963). 6. ‘E.. FE. Necheles, (1961). 7. T.. Kamei, R. Michel, J. Roche, Rend. Soc. Biol. 156, 1236 (1962). 8. R. W. Hanson, R. H. Lindsay, S. B. Barker, Biochim. Biophys. Acta 68, 134 (1963). 9. O. Stein and J. Gross, Proc. Soc. Exptl. Biol. Med. 109, 817 (1962). 10. J. R. Tata, L. Ernster, O. Lindberg, Nature 193, 1058 (1962); J. R. Tata, L. Ernster, O. Lindberg, E. Arrhenius, S. Pedersen, R. Hedman, Biochem. J. 86, 408 (1963). 11 A. San Pietro and D. Rittenberg, J. Biol. Chem. 201, 457 (1953). 12. C. B. Klee, J. Cason, L. Sokoloff, Federation Proc. 22, 580 (1963). 13. M. A. Spirtes, G. Medes, S. Weinhouse, J. Biol. Chem. 204, 705 (1953). 14. O. Ferrini, G. L. Perroni, M. Bestagno, Minerva Nucleare 3, 210 (1959). 15. F. Friedberg and D. M. Greenberg, J. Biol. Chem. 168, 405 (1947); E. Roberts and D. G. Simonsen, in Amino Acid Pools, J. T. Hold- en, Ed. (Elsevier, Amsterdam, 1962), pp. 343-346. 16. E. S. Gordon and A. E. Heming, Endocri- nology 34, 353 (1944). 17. W. P. Weiss and L. Sokoloff, Science 140, 1324 (1963). 17 May 1963 Science 129, we Federation Proc. 20, 67 Compt.