Gene transcription in differentiating immature T cell receptor(neg) thymocytes resembles antigen-activated mature T cells.

Early in ontogeny thymocytes have a surface marker phenotype that resembles activated mature T cells but they lack expression of the T cell receptor (TCR) complex. We have made preparations of day 14/15 triple negative fetal thymocytes that exhibit the activated T lymphocyte markers CD25, intercellular adhesion molecule 1, Ly-6A/E, CD44, and heat stable antigen and are rapidly proliferating as evidenced by flow cytometric examination of BrdU incorporation. We found that binding activities of the gene regulators nuclear factor (NF)-kappa B, the NF-kappa B p50 homodimer complex, nuclear factor of activated T cells (NF-AT), oct-1, oct-2, activator protein 1 (AP-1), and serum response factor (SRF), are all present in these early thymocytes. Whereas the octamer factors and SRF persist during ontogeny, NF-kappa B, NF-AT, and AP-1 decrease and are undetectable in the adult thymus. Transfection of disaggregated thymocytes by electroporation or intact thymic lobes by gold-particle bombardment revealed that reporter constructs for NF-kappa B, NF-AT, AP-1, octamer factors and, to a small extent, the TCR-alpha enhancer were active in early thymocyte development. We rigorously eliminated the possibility that these transcriptional events were due to minor populations of TCR+ cells by showing that these reporter constructs were also active in recombinase activating gene (RAG)-/- thymocytes that are incapable of completing TCR gene rearrangement, and predominantly contain cells that have an activated phenotype. Thus, transcriptional events that are usually triggered by antigen stimulation in mature T cells take place early in thymic ontogeny in the absence of the TCR. Our analysis suggests that there are striking regulatory similarities but also important differences between the activation processes that take place in antigen-stimulated mature T cells and thymic progenitor cells.

Specific triiodothyronine binding sites in the anterior pituitary of the rat.

Studies with L-[(125)l] triiodothyronine and L-[(125)l] thyroxine, and with equilibrium dialysis of plasma proteins indicate that rat pituitary binds L-triiodothyronine 9.8 times as strongly as it does L-thyroxine. Injection of even small doses of nonradioactive L-triiodothyronine reduces the pituitary/ plasma ratio of radioactive L-triiodothyronine, an indication of the existence of pituitary binding sites with a limited capacity for L-triiodothyronine. Limited capacity binding sites for L-thyroxine could not be demonstrated.

Stereospecific transport of triiodothyronine from plasma to cytosol and from cytosol to nucleus in rat liver, kidney, brain, and heart.

We have investigated the transport of L- and D-triiodothyronine (T3) from plasma to cellular cytoplasm and from cytoplasm to nucleus by estimating the concentration of free hormone in these compartments in rat liver, kidney, brain, and heart. We assessed the distribution of T3 in various tissues and its metabolism by standard isotopic techniques and measured plasma and cytosolic tissue T3 by radioimmunoassay. In addition, we determined the fraction of radiosensitive T3 associated with the cytosol in individual tissues and estimated the cytosolic volume per gram of tissue. Equilibrium dialysis allowed us to determine the binding power of cytosols and plasma, and in vitro saturation techniques provided values for the affinity (ka) for L- and D-T3 of isolated nuclei in aqueous solution at 37 degrees C. We calculated the free cytosolic hormone from the product of cytosolic T3 and the binding power of cytosol for T3, and the free intranuclear T3 from the ka and previously determined ratio of occupied-to-unoccupied binding sites under steady state conditions in euthyroid animals. Our results showed that the free cytosolic/free plasma concentrations for L-T3 and D-T3, respectively, were: liver 2.8, 21.6; kidney 1.17, 63.3; heart 1.31, 1.58; brain 0.86, 0.24. The free nuclear/free cytosolic ratios for L-T3 and D-T3, respectively, were: liver 58.2, 3.70; kidney 55.9, 1.54; heart 80.6, 24.9; and brain 251, 108.6. Our findings suggest that stereospecific transport occurs both from plasma to cytosol and from cytosol to nucleus. The high gradients from cytosol to nucleus imply that there is an energy-dependent process and appear to account for the differences in the nuclear association constant determined in vivo and in vitro.

Tissue iodoprotein formation during the peripheral metabolism of the thyroid hormones.

The formation of tissue iodoproteins during the peripheral metabolism of the thyroid hormones was examined by determining the concentration of nonethanol-extractable (125)I (NE(125)I) in various tissues after the intravenous injection of 3,5,3'-triiodo-L-thyronine (T3-(125)I) and L-thyroxine-(125)I (T4-(125)I) in groups of rats with iodide-blocked thyroid glands. 3 days after T3-(125)I and 7 days after T4-(125)I injection the concentration of NE(125)I in the liver and kidney was 5-10 times greater than in plasma. Smaller but nonetheless significant concentrations of NE(125)I were demonstrated in skeletal and cardiac muscle. Hepatic subcellular fractionation studies revealed that the major portion of the liver NE(125)I was in the microsomal fraction. Lower concentrations of NE(125)I were present in the nuclear, mitochondrial, and soluble fractions. When similar studies were performed in groups of rats pretreated with phenobarbital, an increase in the metabolic clearance of T3-(125)I (30%) and T4-(125)I (100%) was observed along with a highly significant increase in the NE(125)I concentration of the liver and plasma. The increase in hepatic NE(125)I in these studies was primarily due to the microsomal component. Incubation of hepatic microsomes with T3-(125)I and T4-(125)I showed that NEI formation as well as deiodination appeared to obey simple Michaelis-Menten kinetics. Moreover, the maximal rate of both deiodination and NEI formation was increased when microsomes harvested from phenobarbital-treated rats were employed. These studies indicate that thyroid hormone metabolism results in the formation of structural and soluble tissue iodoproteins in addition to circulating iodoproteins. The rate of formation of these moieties in the liver and plasma appears to be related to the rate of hormone metabolism.

Propylthiouracil inhibits the conversion of L-thyroxine to L-triiodothyronine. An explanation of the antithyroxine effect of propylthiouracil and evidence supporting the concept that triiodothyronine is the active thyroid hormone.

6-n-propylthiouracil (PTU) administered to male Sprague-Dawley rats maintained on 2 and 5 mug L-thyroxine (T(4))/100 g body weight resulted in a marked reduction in the rate of conversion of L-thyroxine to L-triiodothyronine (T(3)). These effects could not be ascribed to induced hypothyroidism since the group maintained on 5 mug T(4)/day had normal levels of liver mitochondrial alpha glycerophosphate dehydrogenase. In confirmation of previous studies, PTU also reduced the fractional rate of deiodination of T(3). These observations provide a possible explanation of the many published observations indicating that PTU antagonizes the tissue effects of T(4) but not of T(3). The data suggest that monodeiodination of T(4) but not of T(3) is essential before hormonal effects can be manifested at the cellular level.

Quantitation of extrathyroidal conversion of L-thyroxine to 3,5,3'-triiodo-L-thyronine in the rat.

Studies of the rate of extrathyroidal conversion of thyroxine (T4) to 3,5,3'-triiodo-L-thyronine (T3) were carried out in rats. Total body homogenates were prepared and extracted with ethanol 48, 72, and 96 hr after the intravenous injection of (125)I-T4. (131)I-T3 was added, and the paper chromatographic purification of T3 was effected by serial elution and rechromatography in three paper and one thin-layer cycles. The ratio of (131)I-T3 and (125)I-T3 counting rates in the final chromatograms, which was identical in three different paper chromatography systems, was used to calculate the proportion of (125)I-T3 to (125)I-T4 in the original homogenates. In order to discount the effects of in vitro monodeiodination of T4 during extraction and chromatography, we killed control animals immediately after injection of (125)I-T4 and processed them in a similar fashion to the experimental groups. The average ratio of (125)I-T3 to (125)I-T4 in carcass extracts of animals killed between 48 and 96 hr after isotopic injection was 0.08 whereas the average ratio of (125)I-T3 to (125)I-T4 in chromatograms of control animals was 0.01. On the basis of the proposed model, calculations indicated that about 17% of the secreted T4 was converted to T3. Assuming values cited in the literature for the concentration of nonradioactive T3 in rat plasma, these findings would suggest that about 20% of total body T3 is derived by conversion from T4. Moreover, since previous estimates have suggested that in the rat, T3 has about 3 to 5 times greater biologic activity than T4, these results also raise the possibility that the hormonal activity of T4 may be dependent in large part on its conversion to T3.A necessary assumption in calculating T4 to T3 conversion in this and other studies is that the 3' and 5' positions are randomly labeled with radioiodine in phenolic-ring iodine-labeled T4. Evidence supporting this assumption was obtained in the rat by comparing the amount of labeled T3 produced after injection of phenolic and nonphenolic-ring iodine-labeled T4.

Slow fractional removal of nonextractable iodine from rat tissue after injection of labeled L-thyroxine and 3,5,3'-triiodo-L-thyronine. A possible clue to the mechanism of initiation and persistence of hormonal action.

Previous studies have shown that a small but significant proportion of radioiodine from labeled L-thyroxine (T(4)) and 3,5,3'-triiodo-L-thyronine (T(3)) is incorporated into plasma and tissue proteins and is not, therefore, extractable with ethanol or other organic solvents. Other studies have shown that the complex consists, at least in part, of the iodothyronine in apparent covalent linkage with protein. In the present series of experiments the disappearance rate of nonextractable iodine (NEI) was determined in plasma, liver, and kidney after the injection of rats with a single dose of T(4) and T(3) labeled with radioiodine in the phenolic ring. The t(1/2) of NEI decay was substantially longer than the t(1/2) of the initial metabolic removal of T(4) (16 hr) and T(3) (4-6 hr). Thus, between days 3 and 11 the average t(1/2) of plasma NEI derived from T(4) was 2.2 days, from T(3), 1.9 days; kidney NEI from T(4), 7.4 days, from T(3), 6.1 days; hepatic NEI from T(4), 4.3 days, from T(3), 5.2 days. The slow disappearance of liver NEI was of special interest in connection with an analysis of previously published data by Tata and associates dealing with the sequential tissue effects after the injection of a single dose of T(3) into thyroidectomized rats. The t(1/2) of decay of the various biological effects measured, primarily in the liver, appeared similar to each other, averaging between 4 and 6 days. These findings are compatible with the existence of a single long-lived intermediate governing the tissue expression of thyroid hormone. The t(1/2) of hepatic NEI in similarly prepared animals (thyroidectomized and injected with 25 mug of T(3)) was found to be 4.5 days. The coincidence in the slow fractional disappearance rates of hepatic NEI and the dissipation of hormonal tissue effects raises the distinct possibility that T(3) interacts with specific cellular receptor sites to form covalent complexes which are slowly removed and serve both to initiate and to perpetuate hormonal action. A mathematical analysis of hormonal reaction mechanisms, based on the assumption of a linearly responsive system, a t(1/2) of T(3) of 4 hr, and a t(1/2) of 4.5 days for the postulated long-lived "messenger" suggests that maximal expression of hormonal activity cannot be attained before 20 hr after the injection of a hormone pulse. This value is broadly consonant with the observed data accumulated by Tata and associates. The existence of a long-lived messenger, possibly a species of NEI, would therefore explain not only the slow dissipation of hormonal effects but also the well-recognized "lag-time" in the expression of hormonal action.

Decreased serum triiodothyronine in starving rats is due primarily to diminished thyroidal secretion of thyroxine.

Although thyroxine (T4) 5'-deiodinase activity is diminished in liver homogenates of starved rats, no information is available regarding the effect of starvation on net T4 to triiodothyronine (T3) conversion in the intact rat. It appeared important to clarify this relationship since rat liver homogenates are widely used as a model for the study of the factors responsible for reduced circulating T3 in chronically ill and calorically deprived patients. In contrast to the expected selective decrease in circulating T3 levels in calorically restricted humans due to diminished T4 to T3 conversion, 5 d of starvation of two groups of male Sprague-Dawley rats resulted, paradoxically, in a greater decrease in serum T4 than in serum T3 levels. Kinetic studies show that starvation is associated with no change in the metabolic clearance rate (MCR) of T3, a 20% increase in the MCR of T4, a 67% reduction in turnover rate of T4, but only a 58% reduction in the turnover rate of T3. Moreover, in the first group of rats studied, direct chromatographic analysis of the isotopic composition of total body homogenates after the injection of 125I-T4 showed that 21.8% of T4 is converted to T3 in control rats and 28.8% in starved rats, suggesting that virtually all extrathyroidal T3 in starved and control rats is derived from the peripheral conversion of T4, and that there is little or no direct thyroidal secretion of T3. Our findings strongly point to a reduced thyroidal secretion of T4 as the primary cause of the observed reduction in circulating T3. Since the mechanisms leading to reduced levels of plasma T3 differ in humans and rats, it may be important to reexamine the use of liver homogenate preparations as models for study of the pathogenesis of the "low T3 syndrome" in humans.

Relationship between the accumulation of pituitary growth hormone and nuclear occupancy by triiodothyronine in the rat.

Studies were undertaken in hypothyroid rats in an effort to define the kinetics of growth hormone (GH) accumulation in response to i.v. pulse injections of triiodothyronine (T(3)) and to calculate the relationship between nuclear occupancy by T(3) and the instantaneous rate of accumulation of pituitary GH. Results were contrasted to the findings in previous studies of the induction of hepatic mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and malic enzyme (ME) by T(3). The dose of T(3) required to achieve half-maximal accumulation of GH in 24 h was 0.6 mug/100 g body wt, a value 15-fold less than the half-maximal dose for alpha-GPD and ME induction at a comparable time after injection. Although significant increase in pituitary GH were evident as early as 3 h after injection of maximally effective doses of T(3), the rate of increase became linear only 12 h after injection. After achievement of peak values, the pituitary content of GH decayed with a similar terminal t((1/2)) of 3.9 days and 4.1 days in two groups of animals injected with a single dose of 1.0 and 50 mug T(3)/100 g body wt, respectively. In vivo isotopic displacement studies carried out at the equilibrium time point indicated that the pituitary nuclear binding capacity was 5.5 ng T(3)/g tissue and that the plasma concentration at which one-half of the nuclear sites are occupied is 1.0 ng/ml. Nuclear occupancy as a function of time was calculated from the estimated plasma T(3) concentration after injection of the dose and the half-occupancy plasma concentration. These data were then analyzed by application of the mathematical model previously developed to ascertain the relationship between nuclear occupancy and the rate of hepatic enzyme induction. Results indicated that the pituitary nuclear occupancy-response relationship was generally linear, in marked contrast to the highly amplified relationship between nuclear occupancy and the response of ME and alpha-GPD to T(3) in the liver. In supplementary experiments, euthyroid rats received daily injections of 200 mug of T(3) for 7 days to keep nuclear sites nearly saturated for the duration of the experiment. No significant increase in the pituitary GH content above euthyroid base-line levels was noted. This also contrasts with the marked increase above euthyroid levels in alpha-GPD and ME observed in previous studies. Our findings suggest the existence of major differences between the specific mechanisms which lead to the induction of pituitary GH and the hepatic enzymes by T(3).

Functional relationship of thyroid hormone-induced lipogenesis, lipolysis, and thermogenesis in the rat.

Metabolic balance studies were carried out to determine the interrelationships of thyroid hormone-induced lipogenesis, lipolysis, and energy balance in the free-living rat. Intraperitoneal doses of 15 micrograms triiodothyronine (T3)/100 g body wt per d caused an increase in caloric intake from 26.5 +/- 1.7 (mean +/- SEM) kcal/100 g per d to 38.1 +/- 1.5 kcal/100 g per d. Food intake, however, rose only after 4-6 d of treatment and was maximal by the 8th day. In contrast, total body basal oxygen consumption rose by 24 h and reached a maximum by 4 d. Since total urinary nitrogen excretion and hepatic phosphoenolpyruvate carboxykinase mRNA did not rise, gluconeogenesis from protein sources did not supply the needed substrate for the early increase in calorigenesis. Total body fat stores fell approximately 50% by the 6th day of treatment and could account for the entire increase in caloric expenditure during the initial period of T3 treatment. Total body lipogenesis increased within 1 d and reached a plateau 4-5 d after the start of T3 treatment. 15-19% of the increased caloric intake was channeled through lipogenesis, assuming glucose to be the sole substrate for lipogenesis. The metabolic cost of the increased lipogenesis, however, accounted for only 3-4% of the T3-induced increase in calorigenesis. These results suggest that fatty acids derived from adipose tissue are the primary source of substrate for thyroid hormone-induced calorigenesis and that the early increase in lipogenesis serves simply to maintain fat stores. Since the mRNAs coding for lipogenic enzymes rise many hours before oxygen consumption and lipolysis, these results suggest that T3 acts at least in part by an early coordinate induction of the genes responsible for these processes.

Opposing effects of glucagon and triiodothyronine on the hepatic levels of messenger ribonucleic acid S14 and the dependence of such effects on circadian factors.

We have studied the effect of glucagon on the expression of a triiodothyronine (T3) and carbohydrate-inducible mRNA sequence (mRNA-S14) in rat liver that undergoes a threefold diurnal variation (peak, 2200 h; nadir, 0800 h). Glucagon injection into euthyroid rats (25 micrograms/100 g body wt i.p., three doses at 15-min intervals) during the nocturnal plateau of mRNA-S14 caused a monoexponential disappearance of this sequence (t1/2, 90 min) accompanied by a 90% reduction in the transcriptional rate in a nuclear run-off assay, indicative of a near total reduction of synthesis. This effect was markedly attenuated in rats treated with T3 (200 micrograms/100 g body wt i.p.) 24 h before glucagon injection. When T3 was given 15 min after glucagon, the glucagon-initiated decline in mRNA-S14 was reversed within 90 min, suggesting a rapid interaction between the two hormones in the evening. Curiously, administration of T3 alone at this hour did not affect a significant increase in mRNA-S14. At 0800 h, however, T3 caused the expected brisk induction of this sequence, whereas glucagon was without effect. In essence, glucagon affected mRNA-S14 synthesis only in the evening, while T3 increased levels of this sequence above the baseline only in the morning. T3, however, reversed the effect of prior glucagon injection at night. The observed alterations in hormonal responsivity could underly the diurnal variation of mRNA-S14 expression. Moreover, the data suggest the hypothesis that T3 may act on S14 gene expression by antagonizing factors that inhibit its transcription.

Limited binding capacity sites for L-triiodothyronine in rat liver nuclei. Nuclear-cytoplasmic interrelation, binding constants, and cross-reactivity with L-thyroxine.

Further studies have been performed to define the kinetic characteristics of nuclear triiodothyronine (T(3)) binding sites in rat liver (J. Clin. Endocrinol. Metab. 1972. 35: 330). Sequential determination of labeled T(3) associated with nuclei and cytoplasm over a 4-h period allowed analysis of the relationship of T(3) in nuclear and cytoplasmic compartments. A rapid interchange of hormone between nuclei and cytoplasm was demonstrated, and in vitro incubation experiments with nuclei yielded no evidence favoring metabolic transformation of T(3) by the nuclei. In vivo displacement experiments were performed by subcellular fractionation of liver (1/2) h after injection of [(125)I]T(3) with increasing quantities of unlabeled T(3). The nuclear binding capacity for T(3) could be defined (0.52 ng/mg DNA). Analysis of these experiments also allowed an estimation of the association constant of nuclear sites for T(3) (4.7 x 10(11)M(-1)). The affinity of these sites for T(3) was estimated to be 20-40 fold greater than for thyroxine (T(4)). Chromatographic analysis of the nuclear radioactivity after injection of labeled T(4) indicated that the binding of T(4) by the nucleus could not be attributed to in vivo conversion of T(4) to T(3) but reflected intrinsic cross-reactivity of the two iodothyronines at the nuclear binding sites.

Nonlinear (amplified) relationship between nuclear occupancy by triiodothyronine and the appearance rate of hepatic alpha-glycerophosphate dehydrogenase and malic enzyme in the rat.

Three separate approaches were applied to examine the general relationship between R, the rate of induction of specific enzymes (mitochondrial alpha-glycero-phosphate dehydrogenase and cytosolic malic enzyme) and q, the fractional nuclear occupancy by triiodothyronine in male Sprague-Dawley rats. Daily 200-microgram injections of triiodothyronine per 10u g body wt for 7 days resulted in saturation of the hepatic nuclear sites and the achievement of an apparent new steady state of enzyme levels. The increase achieved over base-line hypothyroid levels was then compared with the increment over hypothyroid base line characteristic of intact euthyroid animals with 47% of nuclear sites occupied. The maximal theoretical reate of steady-state enzyme induction could be protected on the basis of the observed maximal increase in enzyme activity observed 1 day after the injection of graded doses of hormone and lambda, the known fractional rate of enzyme dissipation. The 24-h dose-response studies were used to generate R as a continuous function of q, both in hypothyroid as well as in euthyroid animals. This approach involved the numerical solution of an ordinary differential equation describing the rate of change of enzyme as a function of R, which was assumed to be uniquely related to q. Results of these analyses indicated that the ratio of the maximal rate of induction of enzyme at full occupancy to the rate of induction under euthyroid conditions assumes a value between 9.0 and 19.5, depending on the precise analytic and experimental approach applied. This value is far in excess of the theoretical ratio 2.13 which on would anticipate if R were linearly related to q and 47% of the nuclear sites occupied under physiological conditions. Thus, the signal for enzyme induction appears to undergo progressjive amplification with increasing nuclear occupancy. Moreover, the curve describing the relationship between R and q appears highly nonlinear throughout (concave upwards). Although the molecular mechanism responsible for amplification is unknown, recognition of this phenomenon may be helpful in understanding tissue effects of thyroid hormone excess. Moreover, the analytic technique for determining R as a function of q may be of general applicability in studying hormonal response systems under nonsteady-state conditions.

Stimulation of hepatic mitochondrial alpha-glycerophosphate dehydrogenase and malic enzyme by L-triiodothyronine. Characteristics of the response with specific nuclear thyroid hormone binding sites fully saturated.

Experiments were designed to analyze the relationship of a single i.v. dose of triiodothyronine (T3), the level of plasma and hepatic nuclear T3 attained, and the tissue response as reflected in increased activity of hepatic mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytosol "malic enzyme" (ME). These studied were carried out in euthyroid rats by varying the dose of T3 injected and the time at which the animals were killed and the enzyme levels measured. The plasma T3 concentration was determined and the fraction of nuclear sites occupied at any time t was calculated from the known plasma:nuclear relationship. As a first step, the analysis was confined to the limiting situation in which all nuclear sites were effectively saturated. The following additional information was required and obtained: A proportional relationship between the half-neutralizing volume of a specific antiserum to malic enzyme and the activity of malic enzyme was established, thus confirming previous reports that the increase in enzyme activity induced by T3 is due to increased enzyme mass. The absolute refractory period immediately after i.v. injection of T3, during which no enzyme response could be detected, was determined. This was shown to be 13.4 h for alpha-GPD and 8.2 h for ME. Lastly, the t1/2 of the enzyme decay after pulse injection of T3 was measured. This was similar for both enzymes, 2.8+/-0.6 (SD) days for alpha-GPD and 2.7+/-0.6 (SD) days for ME. The results of these studies indicated that the extent of hepatic response appears limited by full occupancy of a set of intracellular receptor sites by T3 which is in rapid equilibrium with the plasma hormone pool. The kinetic properties of the receptors, as functionally defined in these studies, resemble those associated with the recently described specific nuclear T3 sites. These data per se are thus compatible with but do not prove a nuclear site of initiation of hormone effect. Thye do allow the development of an internally consistent mathematical model which permits prediction of enzyme response when the receptor sites are fully occupied for a given length of time after the i.v. injection of hormone. A separate series of studies was carried out in thyroidectomized rats. The response characteristics of alpha-GPD were similar to those observed in euthyroid animals. In contrast, however, the early response of ME to pulse injections of T3 was very much reduced in hypothyroid animals as compared to euthryoid animals in which nuclear sites were saturated for comparable periods. These findings raise the possibility that a factor required for the induction of malic enzyme but not alpha-GPD is deficient in the hypothyroid state.

Glucose and triiodothyronine both induce malic enzyme in the rat hepatocyte culture: evidence that triiodothyronine multiplies a primary glucose-generated signal.

We have stimulated in a cultured hepatocyte system the synergistic interaction between triiodothyronine (T3) and dietary carbohydrate in the induction of malic enzyme (ME). Kinetic studies revealed that isolated hepatocytes equilibrate with media T3 within 5 min; nuclei equilibrate with media T3 by 45 min; and the half-time of T3 metabolism was 10 h in 10% serum. We demonstrated nuclear T3 receptors in isolated hepatocytes and the induction of ME by T3 in physiological concentrations. However, in the complete absence of T3 glucose could still induce ME. At all concentrations of glucose (100-1,000 mg/dl), T3 (0.3 nM free T3) resulted in a relatively constant (1.4- to 1.7-fold) increase in ME response. The augmentation in ME activity was paralleled by an enhanced rate of enzyme synthesis as determined by [3H]leucine incorporation into immunoprecipitable ME. Cells cultured in serum free media also demonstrated a glucose-dependent increase in ME. Insulin greatly stimulated the glucose induction of ME, whereas dexamethasone had very little influence on ME induction. These studies demonstrate the usefulness of an adult hepatocyte tissue culture model for the study of the effects of T3 on gene expression in cells that are not derived from tumor. They clearly demonstrate that well established effects of T3 can be simulated in such a system at levels of free hormone that approximate those in extracellular body fluids. Our results indicate that an increased concentration of glucose per se can induce the formation of ME in the absence of alterations in extrahepatic hormones or factors. Moreover, our findings confirm inferences from in vivo studies that T3 acts as a multiplier of a glucose-induced signal.

Synergism of thyroid hormone and high carbohydrate diet in the induction of lipogenic enzymes in the rat. Mechanisms and implications.

We have investigated the relationship between the administration of triiodothyronine (T3) and a high carbohydrate (CHO) fat-free diet in the induction of lipogenic enzymes in two rat tissues, liver, and fat. Male thyroidectomized rats were treated with graded daily doses of T3 and either supplemented with a high CHO diet or left on a regular diet. Enzymes studied included malic enzyme (ME), fatty acid synthetase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. In the liver, all four lipogenic enzymes showed a synergistic response between T3 administration and high CHO feeding. In fat, ME also responded synergistically. The interaction was reflected in an increased sensitivity to T3. The dose of T3 required to achieve 50% maximal response was reduced three- to seven-fold by the high CHO diet. This phenomenon could not be attributed to a dietary-induced alteration either in T3 metabolism or in number or affinity of the T3-nuclear receptors. Moreover, studies of the relative rate of synthesis of ME suggested a simultaneous time of onset in the induction of ME, within 2 h after the application of either T3 or CHO. Thus, it is unlikely that either stimulus is secondary to the other. Since parallel experiments from this laboratory (Towle, Mariash, and Oppenheimer,1980. Changes in hepatic levels of messenger ribonucleic acid for malic enzyme during induction by thyroid hormone or diet. Biochemistry. 19: 579-585.) show that ME induction both by CHO and T3 is mediated by an increase in specific messenger RNA for ME, the interaction of T3 and the dietary factor occurs at a pretanslational level.

Differences in primary cellular factors influencing the metabolism and distribution of 3,5,3'-L-triiodothyronine and L-thyroxine.

Administration of phenobarbital, which acts exclusively on cellular sites, results in an augmentation of the liver/plasma concentration ratio of L-thyroxine (T4) in rats but no change in the liver/plasma concentration ratio of L-triiodothyronine (T3). Whereas phenobarbital stimulates the fecal clearance rate both of T3 and T4, it increases the deiodinative clearance rate of T4 only. These findings suggest basic differences in the cellular metabolism of T3 and T4. Further evidence pointing to cellular differences was obtained from a comparison of the distribution and metabolism of these hormones with appropriate corrections for the effect of differential plasma binding. The percentage of total exchangeable cellular T4 within the liver (28.5) is significantly greater than the corresponding percentage of exchangeable cellular T3 within this organ (12.3). Extrahepatic tissues bind T3 twice as firmly as T4. The cellular metabolic clearance rate (= free hormone clearance rate) of T3 exceeds that of T4 by a factor 1.8 in the rat. The corresponding ratio in man, 2.4, was determined by noncompartmental analysis of turnover studies in four individuals after the simultaneous injection of T4-(125)I and T3-(131)I. The greater cellular metabolic clearance rate of T3 both in rat and man may be related to the higher specific hormonal potency of this iodothyronine.

Starvation and hypothyroidism exert an overlapping influence on rat hepatic messenger RNA activity profiles.

To assess the effect of starvation and to explore the potential interrelationship of starvation and thyroid status at the pretranslational level, we have analyzed by two-dimensional gel electrophoresis, the hepatic translational products of starved and fed euthyroid and hypothyroid rats. 5 d of starvation resulted in a statistically significant change in 27 of 240 products visualized, whereas hypothyroidism caused a change in 20, both in comparison with the fed euthyroid state. Of considerable interest was that 68% of all changing messenger (m)RNA sequences were common to the hypothyroid and starved groups and showed the same directional shift. Further, both starvation and hypothyroidism yielded comparable decreases in total hepatic cytoplasmic RNA content. Although it has been well established that the level of circulating triiodothyronine (T3) and the level of hepatic nuclear receptors fall in starvation, this reduction cannot account for the observed decrease of total hepatic RNA nor for all of the alterations in the concentrations of specific mRNA sequences. Thus, administration of T3 to starved animals in a dose designed to occupy all nuclear T3 receptors fails to prevent the fall in total RNA and the majority of starvation-induced changes in the level of mRNA sequences. Moreover, starvation of athyreotic animals results in a further decrease in total RNA and in a further change in the level of individual mRNA species. We conclude, therefore, that although the reduced levels of circulating T3 and the nuclear T3 receptors can contribute to the observed results of starvation, the starvation-induced changes are not exclusively mediated by this factor. The striking overlap in the genomic response between hypothyroid and starved animals raises the possibility that those biochemical mechanisms regulated at a pretranslational level by T3 are either not helpful or injurious to the starving animal. The reduction in circulating T3 and nuclear receptor sites together with T3-independent mechanisms initiated in the starved animal may constitute redundant processes designed to conserve energy and substrate in the nutritionally deprived organism.

Thyroid hormone-carbohydrate interaction in the rat: correlation between age-related reductions in the inducibility of hepatic malic enzyme by triiodo-L-thyronine and a high carbohydrate, fat-free diet.

Previous studies from this laboratory have demonstrated an age-related decrease in hepatic malic enzyme (ME) levels and in the response of ME to triiodo-l-thyronine (T(3)). Moreover, we have recently shown a synergistic interaction of T(3) and a high carbohydrate diet in the induction of this enzyme. Studies were therefore undertaken to assess the response of aging rats to a high carbohydrate diet and to test the effect of such dietary manipulations on the responsiveness of ME to T(3). For this purpose, a new radio-immunoassay for ME was developed that, because of a 10-fold higher sensitivity, was particularly suited to the measurement of the low concentrations of hepatic enzyme in older animals. The level of ME per milligram of DNA fell approximately 70% between 1 and 6 mo with only minor further changes demonstrated between 6 and 18 mo. In contrast, the level of ME per milligram DNA in brain was slightly increased in the older animals. Although the absolute increment of hepatic ME resulting from seven daily injections of T(3) (15 mug/100 g body wt) fell with age, the ratio of the ME content per milligram DNA to that observed in control animals maintained on a regular chow diet remained relatively constant with an average value of 11.1. The responsivity of hepatic ME to a high carbohydrate, fat-free diet also decreased with age and could not be attributed exclusively to a reduction in food consumption. The age-related reduction in ME responsivity to dietary stimuli appeared to be due to a reduction in the formation of the specific messenger, (m)RNA for ME as determined in an in vitro translational assay. Our data are consistent with the following hypothesis. There is an age-related decreased hepatic responsivity to a high carbohydrate dietary stimulus. Thyroid hormone administration, as previously postulated by us, interacts with a product or an intermediate of carbohydrate metabolism in a multiplicative fashion. As a consequence, the absolute increment of ME induced by T(3) administration also declines with age.

Hepatic messenger ribonucleic acid activity profiles in experimental azotemia in the rat. Relationship to food intake and thyroid function.

We have studied the hepatic messenger RNA (mRNA) activity profile in chronically azotemic rats and sought to determine whether the observed changes could be mediated either by reduced food intake or diminished thyroid function at the tissue level. mRNA activity profiles were produced by two-dimensional gel electrophoretic separation of radioactively labeled products of an in vitro reticulocyte lysate system which had been programmed by hepatic RNA. Of the approximately 240 translational products identified in this system, seven sequences were consistently altered in azotemia. In pair-fed animals six of these also decreased, but the alterations in three were depressed to a significantly lesser extent in the pair-fed group. Moreover, analysis of covariance suggested that food intake could account for the differences in only one sequence. The possibility that the mRNA activity profile in azotemia could represent the effects of diminished thyroid function was minimized by the finding that the reductions in plasma thyroxine (T4) and triiodothyronine (T3) levels observed were due largely to reduced plasma protein binding, with maintenance of the mean free T4 and free T3 concentrations within the normal range. The changes in only one mRNA sequence could be related to free T3 levels alone. Our findings, therefore, indicate that although diminished food intake and reduced thyroid function may contribute to some of the observed changes in the mRNA activity profiles, the bulk of alterations in azotemia appear to be mediated by other mechanisms. The striking overlap between the sequences affected by azotemia and pair-feeding raises the speculation that altered gene expression in azotemia may reflect an impaired hepatic response at the pretranslational level to metabolic signals associated with food intake.