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THE MITOCHONDRIAL ROUTE OF THYROID HORMONE ACTION* KENNETH STERLING, M. D. Clinical Professor of Medicine Columbia University College of Physicians and Surgeons New York, N.Y. Director, Protein Research Laboratory Veterans Administration Hospital Bronx, N.Y.

T HE mechanism of action of thyroid hormones upon the peripheral tissues of the body is an intriguing subject which has engaged the attention of a number of eminent investigators. Some suggested mechanisms, all supported by definite evidence, are listed in Table I. In view of all the findings which have been accumulated, the seventh item, namely, combinations of two or more pathways, seems the most reasonable. Drs. Herbert H. Samuels' and Jack H. Oppenheimer2 have presented in detail the evidence for action at the level of the cell nucleus by increasing the transcription of the genetic message and the formation of increased messenger RNA (mRNA) directing the synthesis of hormonally induced proteins. This concept bears a general similarity to the widely accepted model for steroid hormone action which is illustrated in Figure 1, based upon extensive studies of estrogens, androgens, progesterone, aldosterone, and glucocorticoids.'~ In contrast to the foregoing, we propose a dual action of the thyroid hormones, illustrated in Figure 2. This shows the active hormone triiodothyronine (T3) interacting with the effector loci of the mitochondria as well as the nucleus. We have been studying the interaction of labeled thyroid hormones with the subcellular compartments, the cytosol, the nucleus, and the mitochondria in an effort to gain information with regard to the proteins which bind thyroid hormones. *Presented as part of a Symposium on the Thyroid held by the Section on Medicine of the New York Academy of Medicine November 20, 1975. This research was supported in part by Grant AM 10739 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Md., and by the Veterans Administration Medical Research Service, Washington, D.C. Address for reprint requests: V.A. Hospital, 130 West Kingsbridge Road, Bronx, N.Y. 10468.

Bull. N.Y. Acad. Med.

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ACTION261

TABLE I. SUGGESTED MECHANISMS OF ACTION OF THYROID HORMONE 1) Nuclear transcription 2) Mitochondrial activation 3) Na-K ATPase ("sodium pump") 4). Incorporation into tyrosine pathways 5) Adrenergic receptor sensitivity 6) Membrane action 7) Combinations of the above mechanisms

Fig. 1. Model for steroid hormone action on target cell. The sequence of events depicted has been verified to some extent for the following steroid hormones: estradiol, testosterone, progesterone, cortisol and analogues, and aldosterone. The pentagon designated H represents the steroid hormone molecule. The cytosol receptor protein is designated receptor; change in shape signifies an apparent change in protein conformation or dimerization. mRNA = messenger RNA. Vol. 53, No. 3, April 1977

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Fig. 2. Model for thyroid hormone action on target cell. The sequence of events is believed to be similar for thyroxine (T4) and triiodothyronine (T3). In the model illustrated, T3 within a circle indicates the unbound hormone, which diffuses into the cell to be bound by the cytosol binding protein (CBP). Rather than being translocated into the nucleus, the CBP-T: complex is believed to be in reversible equilibrium with a minute moiety of intracellular unbound T3 which can interact with the binding proteins of the effector loci, the nucleus and the mitochondria, as shown. We estimate that there are more than 20 million primary sites in the cytosol of rat liver cells; hence, many CBP molecules would not have a bound T3 molecule.

The separations of subcellular compartments were carried out by conventional methods, as described in previous reports.67 The purity of our mitochondrial preparations has been questioned by H. H. Samuels, who asked if these could be contaminated with nuclear fragments; he urged electron micrographic confirmation of our mitochondria. Accordingly, Figures 3 and 4 illustrate electron micrographs of mitochondrial preparations from normal rat liver showing entirely satisfactory pictures. Moreover, the electrophoretic mobilities we have observed for nuclear and mitochondrial binding proteins are so different as to obviate any possibility of confusion. Figure 5 illustrates scans of paper electrophoretic runs on human serum simultaneous with nuclear binding protein from normal rat Bull. N.Y. Acad. Med.

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:'..Am olI.._

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Fig. 5. Thyroid hormone binding protein in kidney nuclei of the rat. Electrophoresis in glycine acetate, pH 8.6. The paper strip of human serum proteins stained with bromphenol blue is illustrated at the top, with the protein bands appropriately labeled, A for albumin and a,, a2, a, and y for the globulins. The small dots at each end of the strip are markers placed prior to scanning. The radioactive scan of human serum with added [1311] T4 was performed prior to the staining of the paper strip. The lower scan of nuclear protein labeled with [1311] T3 may be compared with the scan of human serum which was run simultaneously. In addition to the origin radioactivity, there is a peak with mobility equal to or perhaps slightly greater than thyroxine-binding alpha-globulin (TBG) but slower than albumin (ALB) or prealbumin (TBPA).

kidney. It is readily observed that in addition to an origin peak there is a peak that migrates slightly faster than the mobility of serum thyroxinebinding alpha-globulin (TBG). In contrast, Figure 6 shows paper electrophoretic strips of mitochondrial protein from serum and rat liver run in alkaline sodium borate buffer at pH 10.0 with a major peak for mitochondrial binding protein running with higher mobility than any of the normal human serum proteins. Therefore, it is evident that the mitochondrial protein is different indeed from that which we have obtained from nuclei. These results are typical of a large number of studies on the subcellular proteins obtained from normal and hypothyroid rat liver and kidney preparations. Vol. 53, No. 3, April 1977

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Fig. 6. Thyroid hormone binding protein in mitochondria of rat liver. Electrophoresis was performed oil paper in sodium borate buffer, pH 10.0. The paper strip of human serum proteins stained with bromphenol blue is illustrated at the top, with the protein bands appropriately labeled, A for albumin and a,, a2, a, and y for the globulins. The small dots at each end of the strip were markers placed prior to scanning. The radioactive scan of human serum with added [1311] T4 was performed prior to staining of the paper strip and shows a single large radioactive peak without resolution of prealbumin, albumin, and thyroxine binding alpha-globulin. Lack of resolution of the serum thyroid hormone carriers is usual when serum is run in the strongly alkaline borate buffer system, which has been applied for resolution of subcellular hormone binding proteins. The lower scan of mitochondrial protein represents mitochondrial membrane protein from intact mitochondria that had been incubated with [131I] T3 (5.4 x 10-9 moles/liter.). The protein was applied at the origin, then subjected to electrophoresis. An insignificant radioactive peak is evident at the origin; the major peak has a somewhat higher mobility than that of any of the serum proteins. Reproduced by permission from Sterling, K. and Milch, P.O.: Thyroid hormone binding by a component of mitochondrial membrane. Proc. Nat. Acad. Sci. U.S.A. 72:3225-29, 1975.

Many experiments have suggested that the outer mitochondrial membranes contain nonspecific binding sites which are not saturated readily. Bull. N.Y. Acad. Med.

267

THYROID HORMONE ACTION

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B/F

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kassociation a\

=3.3 x 108 L/M

n=15,000 sites per nucleus

0.05-

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2 B (,uM x 10-4 T3 BOUND)

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Fig. 7. Interaction between triiodothyronine (T3) and rat liver nuclei illustrating saturable, high affinity and low capacity binding sites (Scatchard plot). B/F = bound/free.

This accounts for observations made by our group and others concerning the minimal effect of large loading doses of T3 upon the mitochondrial localization of the hormone in studies carried out with tracer in vivo and in vitro. In other words, one must go beyond the mere isolation of intact mitochondria to find saturable receptors of high affinity and low capacity, which we believe are important in physiological thyroid hormone regulation. An illustration of saturable, high affinity and low capacity interaction between T3 and rat liver nuclei is provided in Figure 7, which resembles the findings we have obtained also with separated nuclear protein. To obtain similar saturable receptors from the mitochondria required at least preliminary fractionation of this organelle. Isolated mitochondria were subjected to sonic disruption, and the membrane fragments were spun down and then treated with Triton X- 100. Such mitochondrial membrane protein showed reproducible saturable binding. Vol. 53, No. 3, Aprl 1977

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Fig. 8. Gel filtration of mitochondrial membrane protein on Sephadex G-200. The calibrations of the 90 x 1.5 cm. column had previously been established with human serum with added [1251]T4, and are indicated in the figure. The initial sharp peak (A) invariably emerged early, approximately one tube after the IgG peak of human serum; hence, it was judged to represent one or more large protein molecules that bind triiodothyronine (T3). The smaller peak (B) usually had its maximum T3 radioactivity and absorbance at about tube 37 or 38 as shown, signifying that a much smaller protein or polypeptide binds T3. Thte mitochondrial membrane protein had been enriched with 5.7 x 10.10 moles/liter [125]T3 prior to gel filtration. Reproduced by permission from Sterling, K. and Milch, P.O.: Thyroid hormone binding by a component of mitochondrial membrane. Proc. Nat. Acad. Sci. U.S.A. 72:3225-29, 1975.

An additional fractionation step of gel filtration on Sephadex G-200, illustrated in Figure 8, yielded a large macromolecule, "A peak," which emerges just one tube after IgG (from prior calibration of the column with human serum). The "B peak" emerged later, signifying molecular size smaller than serum albumin (as illustrated), and this too was found capable of binding thyroxine (T4) and T3. However, the highest association constant for any subcellular material examined to date has been obtained with the A peak from mitochondrial membrane protein. The Scatchard plot illustrated in Figure 9 shows an association constant (K) approximating 3 x 1011 liters/mole. The number of binding sites (n) was derived from the intercept on the abscissa, and calculated as the number of sites per cell based on the value Bull. N.Y. Acad. Med.

269

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B (pM.x I0-6 T3 BOUND) Fig. 9. Interaction between triiodothyronine (T3) and rat liver mitochondrial protein (Scatchard plot). The association constant (K) exceeds 101" liters/mole. The points illustrated were obtained after subtraction of small nonspecific binding obtained from duplicate tubes to which 200 /xg./ml. of T3 had been added (3 x 10-4moles liter). B/F = bound/free.

of 1 x 108 cells per gram (wet weight) of rat liver6'7 and on protein determinations. Our calculations show approximately 2,000 mitochondrial binding sites per liver cell, but this figure is regarded as only a preliminary approximation, since it was computed from data of unfractionated mitochondrial membrane protein, although from a large number of preparations. The values obtained from mitochondria from hypothyroid rats more than a month after thyroidectomy have not differed

significantly. As illustrated in Table II, the association constant for the interaction between T3 and the protein obtained from mitochondrial membrane is of a much greater order of magnitude than that observed for nuclei or cytosol, consistent with our model suggesting earliest action upon mitochondrial energy metabolism. Our data show T3 being bound more firmly than T4 with all the subcellular binding proteins (Table II). The hormone analogues, D-thyroxine (D-T4) and tetraiodothyroacetic Vol. 53, No. 3, April 1977

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TABLE II. ASSOCIATION CONSTANTS (K) FOR THE INTERACTION BETWEEN CELLULAR CONSTITUENTS AND THYROID HORMONES (IN LITERS/MOLE)

T3 Liver cytosol Kidney cytosol Liver nuclei Kidney nuclei Liver mitochondria Kidney mitochondria

T4

2.3 x 106 3.3 x 106 5.0 x 108

TA4

D-T4

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2.0 x 105 8.9 x 105 4.9 x 105 2.0 x 108

1.9 x 1011 9.4 x 10'° 3.3 x 109 2.0 x 1011 6.2 x 1010 5.3 x 109

1.1 X 1010 2.4 x 1012 1.3 x 1010 4.3 x 1012

T3 = triiodothyronine, T4 = thyroxine, TA4 = tetraiodothyroacetic acid, D-T4 = D-thyroxine, and 3 '-isopropyl, 3 .5-T2 = 3 '-isopropyl, 3.5-diiodo-L-thyronine.

acid (TA4), and 3'-isopropyl, 3 ,5-diiodo- L-thyronine (3 '-isopropyl, 3,5- T2) also have been studied with the aid of the equation: 1

A

kB =

-

[B]\ A

1) /

where kA = the association constant for labeled T3 or T4 alone, kA' = the T3 or T4 association constant in the presence of the competing (nonradioactive) ligand, [B] = the concentration of the competing ligand, and kB= the association constant of the competing ligand for the primary site previously determined for labeled T3 or T4 alone. We have used the above equation to study binding by weaker competing ligands for primary binding sites,8 based upon the formulation of Edsall and Wyman,9 for the effects of competition between different ligands for the same binding site. This is illustrated in the Scatchard plot of Figure 10 which depicts the diminution of binding of labeled [1251] T4 by the addition of nonradioactive TA4 as a competing ligand. As shown in Table II, the avidity of interaction with the thyroid hormone analogues was of interest since it bore a self-evident relation to physiological activity; thus, the mitochondrial protein was observed to bind T3 more avidly than T4, as was the case for cytosol and nuclei, whether intact nuclei or the acidic nuclear protein extract was used. The relatively inactive hormone analogues, TA4 and D-T4, were much less firmly bound than the active hormones, T3 and T4. The highly active analogue, 3'-isopropyl, 3,5-T2 had more than tenfold greater affinity for Bull. N.Y. Acad. Med.

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4 lone

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plus TA4 2

3

4

5

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7

B(,uMx10-6T4 BOUND) Fig. 10. Interaction between rat liver mitochondrial protein and thyroxine (T4) and tetraiodothyroacetic acid (TA4) (Scatchard plot). The diminution in binding caused by the presence of stable TA4 at a concentration of 5 x 10-4 umoles/liter is clearly evident. B/F =

bound/free.

the putative receptor than T3, in keeping with its greater physiological activity. The ability of this putative mitochondrial receptor to differentiate between D-T4 and L-T4 was of particular interest. Mitochondria have been obtained by similar methods from the spleen, brain, and testes, and thus far we have been unable to detect specific saturable binding of T3. The tissues from these three organs have been reported to be relatively unresponsive to the usual thyroid hormone effects in increasing Q02 after administration to rats in vivo. 10 Further work is now under way to determine the nature of the mitochondrial membrane protein (MMP) which we conceive to function as an important hormone receptor. At present the evidence available suggests that we are dealing with a thermostable lipoprotein, presumably from the inner mitochondrial membrane. The binding properties are hardly altered by repeated freezing and thawing, in contrast to the well-known lability of serum lipoproteins. The binding studies carried out at 370C. generally have given values similar to those obtained at 00C. with respect to binding constants, stereospecificity, and lesser affinity for inactive hormone analogues. Vol. 53, No. 3, April 1977

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Fig. 11. Disc electrophoretic analysis with polyacrylamide gel, rat kidney mitochondrial membrane protein (MMP). The lipoproteins (L.P.) have been stained with Sudan B which had been added prior to electrophoresis in dilute (3. 1%) gel. The concentrating (conc.) gel is indicated at the left above the resolving gel. The origin and anode are indicated at the right. The first tube at the left represents a normal serum sample with alpha and beta lipoprotein bands (aL.P. and 83L.P.). The second tube is crude MMP and the third and fourth tubes are A and B peaks. The B peak is devoid of lipoprotein; lipoprotein is evident in the A peak as well as in the crude MMP.

Evidence supporting the lipoprotein character of the A peak fraction is provided in Figure 11, in which disc electrophoretic analysis with polyacrylamide gel has been carried out. A serum sample is shown at the left, the second tube is crude MMP and the third and fourth tubes are A and B peaks, all run with a dilute (3. 1 %) gel. This dilute gel permitted lipoprotein staining with Sudan B; the illustration shows clearly the alpha and beta lipoproteins of serum and the lipoproteins of the MMP and A Bull. N.Y. Acad. Med.

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4, 474.X 1=-t-4- T 1TiI1l-z1z24 ¾; A;fLIhIh 1 44 KPL I

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0 2 1 TI ME IN MINUTES Fig. 12. Oxygen consumption by isolated rat liver mitochondria; effect of the thyroid hormone. Representation of an actual tracing with a Clark oximeter carried out on isolated rat liver mitochondria incubated at 370C. in the presence of succinate as substrate. Upon addition of T3 (5 ,umoles/liter), an immediate increase in oxygen consumption (Qo2) is evident. A lesser but detectable effect was observed at a concentration of 0.5 Amoles/liter T3 (500 nanomoles/liter).

peak tubes, but no lipoproteins whatever in the B peak. This implies that a specific lipoprotein receptor in the inner mitochondrial membrane functions as a receptor for the thyroid hormones and may regulate mitochondrial energy metabolism. Further investigations of the physical-chemical characteristics of this putative mitochondrial receptor and its mode of action are urgently needed, and some studies now are under way. (Direct chemical analysis has now confirmed the findings suggested above.) An immediate effect upon the oxygen consumption of isolated rat liver mitochondria is illustrated in Figure 12; it shows a Clark oximeter tracing carried out on isolated rat liver mitochondria incubated at 370C. in the presence of succinate as substrate. The immediate increase in oxygen consumption is evident. However, in the example shown the concentration of T3 employed was 5 ,tmoles/liter, vastly greater than anything encountered in vivo, either in health or in disease. Graded responses have been Vol. 53, No. 3, April 1977

274

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K. STERLING K.

observed, however, extending to lesser concentrations, such as 0.5 /imoles/liter. Of potentially greater significance are current studies'1 employing 32 P as Pi in phosphate buffer in the medium which includes unlabeled adenosine diphosphate (ADP). Intact mitochondria or submitochondrial particles incubated in such a medium incorporate the inorganic phosphate into adenosine triphosphate (ATP). This mitochondrial function of oxidative phosphorylation may be stimulated by thyroid hormones in the physiological range. We have been observing increased incorporation in such incubations on the addition of quite small concentrations of T3, with effects at levels as low as the nanomolar and picomolar ranges, in mitochondria from hypothyroid rats only.1' Evidence has been accumulated which suggests but does not prove that the thyroid hormones may have a direct action upon the mitochondria of responsive cells.12-8 The demonstration of a specific binding protein of high affinity and low capacity as well as stereospecificity in mitochondrial membrane tends to support this viewpoint significantly. The recent evidence from Edelman'9 20 regarding the thermogenic action of sodium-potassium adenosine triphosphatase (ATPase) is fully consistent with our model. The effect upon ATPase is considered a significant final pathway of energy expenditure, although not a likely site of primary thyroid hormone action. Since the association constants herein reported for the putative mitochondrial receptor are of an appreciably higher magnitude than those obtained for the nuclear receptors,21-28 it seems reasonable to postulate that the initial physiologic action of the thyroid hormone is due to a direct stimulation of mitochondrial energy metabolism. This concept in no way excludes the possible role of the nuclear receptors, which are believed to modulate the transcription of the genetic message to increase the synthesis of hormonally induced proteins. This latter effect of thyroid hormone is typified by the role of the hormone in tadpole metamorphosis; the striking changes in growth and development illustrate delayed but sustained effects compatible with the anticipated time lag for the activation of protein synthesis. In summary, the data suggest a dual action of thyroid hormone at the cellular level: 1) an action upon nuclear transcription, mRNA, and protein synthesis-this important hormonal role in growth, differentiation, and cell maintenance is viewed as a sustained action which is neither immediate nor initial; 2) activation of mitochondrial energy metabolism, Bull. N.Y. Acad. Med.

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which is considered a likely candidate for the first effects observable, such as the increased consumption of oxygen demonstrable within hours after intravenous injection of T3 into myxedematous human subjects.29 Indeed, our preliminary experiments with isolated rat liver mitochondria suggest increased ATP formation on addition of T3 in the physiological range. The role of cytosol receptor proteins for thyroid hormones differs strikingly from the postulated models of steroid hormone action. There is no evidence that the thyroid hormones with cytosol binding proteins are "translocated" into the postulated effector loci, the nucleus and mitochondria. Rather, the cytosol proteins may serve a function approximately analogous to that of the binding proteins in the circulating plasma. The cytosol binding proteins may well serve to hold the hormones intracellularly, where they are in equilibrium with a minute moiety of free T3 and free T4 which can, in turn, be bound by the nuclear and mitochondrial receptor proteins. ACKNOWLEDGEMENTS

I am grateful for the collaboration of Mr. Milton A. Brenner and Drs. Peter 0. Milch and John H. Lazarus, and for the critical contributions of Drs. John N. Loeb, Marshall P. Primack, Frederic L. Hoch, and Mrs. Joan Ross Popovitch. I am also indebted to Drs. Fiorenzo Paronetto and Swan Nio Thung for their expert work in preparing electron photomicrographs of isolated mitochondria.

1.

2.

3. 4.

5.,

REFERENCES Samuels, H.H.: In Vitro Studies on tion, its translation in a heterologous Thyroid Hormone Receptor. In: Recepcell-free system, and its control by glucocorticoid hormones. Proc. Natl. tors and Hormone Action, Birnbaumer, L. and O'Malley, B.W., editors. New Acad. Sci. U.S.A. 70:1218-21, 1973. York, Academic, vol. 3. In press. 6. Sterling, K.,Saldanha, V.F., Brenner, Oppenheimer, J.H.: Initiation of M.A., and Milch, P. O.: Cytosol thyroid-hormone action. N. Engl. J. binding protein of thyroxine and Med. 292:1063-68, 1975. triiodothyronine in human and rat kidO'Malley, B.W.: Mechanism of action ney tissue. Nature 250:661-63, 1974. of steroid hormones. N. Engl. J. Med. 7. Sterling, K. and Milch, P.O: Thyroid 285:370-77, 1971. hormone binding by a component of O'Malley, B.W. and Means, A.R.: mitochondrial membrane. Proc. NatI. Female steroid hormones and target Acad. Sci. U.S.A. 72:3225-29, 1975. cell nuclei. Science 183:610-20, 1974. 8. Sterling, K.: Molecular structure of Schutz, G., Beato, M., and Feigelson, thyroxine in relation to its binding by P.: Messenger RNA for hepatic tryphuman serum albumin. J. Clin. Invest. 43:1721-29, 1964. tophan oxygenase: Its partial purifica-

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9. Edsall, J. T. and Wyman J.: BiophysiJ., and Barsano, C.: Nuclear cal Chemistry. New York, Academic, triiodothyronine-binding protein: Partial characterization and binding to 1958, vol. 1, pp. 651-53. 10. Barker, S.B. and Klitgaard, H.M.: chromatin. Proc. Natl. Acad. Sci. Metabolism of tissues excised from U.S.A. 71:4042-46, 1974. thyroxine-injected rats. Am. J. Physiol. 22. DeGroot, L.J. and Torresani, J.: 170:81-86, 1952. Triiodothyronine binding to isolated 11. Sterling, K., Brenner, M.A., and liver cell nuclei. Endocrinology Milch, P. O.: Thyroid hormone action: 96:357-69. 1975. Mitochondrial pathway. In preparation. 23. Oppenheimer, J.H., Koerner, D., 12. Tapley, D. F., Cooper, C., and Schwartz, H.L., and Surks, M.I.: Lehninger, A.L.: The action of Specific nuclear triiodothyronine thyroxine on mitochondria and oxidabinding sites in rat liver and kidney. J. tive phosphorylation. Biochim. Clin. Endocrinol. Metab. 35:330-33, Biophys. Acta 18:597-98, 1955. 1972. 13. Buchanan, J. and Tapley, D.F.: Stim- 24. Surks, M.I., Koerner, D., Dillman, ulation by thyroxine of amino acid inW., and Oppenheimer J.H.: Limited corporation into mitochondria. Endoccapacity binding sites for rinology 79:81-89, 1966. L-triiodothyronine in rat liver nuclei. 14. Primack, M.P., Tapley, D.F., and Localization to the chromatin and parBuchanan, J.: Thyroid hormone stimtial characterization of the ulation of mitochondrial protein L-triiodothyronine-chromatin complex. J. Biol. Chem. 248:7066-72, 1973. synthesis supported by an ATP generating system. Endocrinology 91:840- 25. Surks, M.I., Koerner, D.H., and Op44, 1972. penheimer, J.H.: In vitro binding of 15. Tata, J.R., Ernster, L., and Suranyi, L-triiodothyronine to receptors in rat E. M.: Interaction between thyroid liver nuclei. Kinetics of binding, exhormones and cellular constituents. I. traction properties and lack of requireBinding to isolated sub-cellular partiment for cytosol proteins. J. Clin. Incles and sub-particulate fractions. vest. 55:50-60, 1975. Biochim. Biophys. Acta 60:461-79, 26. Samuels, H.H. and Tsai, J.S.: Thyroid 1962. hormone action in cell culture: De16. Bronk, J.R.: The influence of monstration of nuclear receptors in inthyroxine and related compounds on tact cells and isolated nuclei. Proc. oxidative rate and efficiency of phosNatl. Acad. Sci. U.S.A. 70:3488-92, phorylation in liver mitochondria and 1973. sub-mitochondrial particles. Ann. N.Y. 27. Samuels, H.H., Tsai, J.S., Casanova, Acad. Sci. 86:494-505, 1960. J., and Stanley, F.: Thyroid hormone 17. Bronk, J.R.: Thyroid hormone: Effects action: In vitro characterization of solon electron transport. Science ubilized nuclear receptors from rat liver 153:638-39, 1966. and cultured GHR cells. J. Clin. Invest. 18. Bronk, J.R. and Bronk, M.S.: The in54:853-65, 1974. fluence of thyroxine on oxidative 28. MacLeod, K.M. and Baxter, J.D.: phosphorylation in mitochondria from DNA binding of thyroid hormone rethyroidectomized rats. J. Biol. Chem. ceptors. Biochem. Biophys. Res. 237:897-903, 1962. Commun. 62:577-83, 1975. 19. Edelman, I.S. and Ismail-Beigi, F.: 29. Blackburn, C. M., McConahey, Thyroid thermogenesis and active W.M. Keating, F.R., Jr., and Albert, sodium transport. Rec. Prog. Horm. A.: Calorigenic effects of single inRes. 30:235-57, 1974. travenous doses of L-triiodothyronine 20. Edelman, I.S.: Thyroid thermogenesis. and L-thyroxine in myxedematous perN. Engl. J. Med. 290:1303-08, 1974. sons. J. Clin. Invest. 33:819-24, 1954. 21. DeGroot, L.J., Refetoff, S., Strausser,

Bull. N.Y. Acad. Med.