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Our experiments show that the T3 nonhalogenated analog 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT) exhibits the general activities of thyroid hormones. DIMIT delivered to hypothyroid Rat by constant infusion or sc injections increases bodyweight gain, BMR before or after epinephrine and phosphorylative oxidation of liver mitochondria.
3,5-Dimethyl-3'-isopropyl-L-thyronine (DIMIT, CAS 26384-44-1), a non-halogenated, artificial analogue of the thyroid hormone T3, was administered once daily by intragastric gavage to pregnant Wistar rats on days 17-20 of gestation to elucidate the effects of DIMIT on maternal and foetal thyroid state and gestational outcome under the influence of low iodine or iodine excess nutrition. Doses of 20 micrograms.kg-1.d-1 and 150 micrograms.kg-1.d-1 DIMIT were applied. Throughout pregnancy the animals were kept on low iodine diet and either on iodine free drinking water or iodine excess. There was no statistically significant effect on maternal body weight, weight of living foetus, the rate of intra-uterine death before day 16 of gestation, water and food consumption, and no hint of maternal thyrotoxicosis. The vital off-spring did not reveal any signs of gross malformation. DIMIT decreased serum TSH both in dams and foetus, and diminished the TSH stock of the pituitary gland of the dams. T3-levels were also lowered in maternal serum by DIMIT. These DIMIT induced effects were observed irrespective of iodine deficiency or excess. There was no difference in foetal mortality before onset of DIMIT-treatment, but the gestational death rate increased in a dose-related manner by the administration of DIMIT. This serious adverse effect became apparent at a DIMIT-dose that is not sufficient for a complete compensation of foetal hypothyroidism in utero. The higher prenatal mortality was accompanied by a decreased placental weight, but not by a reduced body weight of surviving foetus.
The thyroid hormone, 3,5,3'-Triiodo-L-thyronine (T3), is essential for growth, differentiation, and regulation of metabolic functions in multicellular organisms, although the specific mechanisms of this control are still unknown. In this study, treatment of a human pancreatic duct cell line (hPANC-1) with T3 blocks cell growth by an increase of cells in G(0)/G(1) cell cycle phase and enhances morphological and functional changes as indicated by the marked increase in the synthesis of insulin and the parallel decrease of the ductal differentiation marker cytokeratin19. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent increase in insulin and glut2 mRNA levels in response to T3. As last step of the acquisition of a beta-cell-like phenotype, we present evidence that thyroid hormones are able to increase the release of insulin into the culture medium. In conclusion, our results suggest, for the first time, that thyroid hormones induce cell cycle perturbations and play an important role in the process of transdifferentiation of a human pancreatic duct line (hPANC-1) into pancreatic-beta-cell-like cells. These findings have important implications in cell-therapy based treatment of diabetes and may provide important insights in the designing of novel therapeutic agents to restore normal glycemia in subjects with diabetes.
To understand the structural basis in the hormone-dependent transcriptional regulation of human beta 1 thyroid hormone receptor (h-TR beta 1), we studied the conformational changes of h-TR beta 1 induced by binding of 3,3',5-triiodo-L-thyronine (T3). h-TR beta 1 was treated with trypsin alone or in the presence of T3, thyroid hormone response element (TRE) or T3 together with TREs. Without T3, h-TR beta 1 was completely digested by trypsin. Binding of TREs had no effect on the tryptic digestion pattern. However, T3-bound h-TR beta 1 became resistant to tryptic digestion and yielded trypsin-resistant peptide fragments with molecular weight of 28,000 and 24,000. Chymotryptic digestion also yielded a T3-protected 24 Kd peptide fragment. Using anti-h-TR beta 1 antibodies and amino acid sequencing, the 28 Kd fragment was identified to be Ser202-Asp456. The 24 Kd tryptic fragments were found to be Lys239-Asp456 and Phe240-Asp456. The 24 Kd chymotryptic fragment was identified to be Lys235-Asp456. The structural changes as a result of T3 binding could serve as a transducing signal to modulate the gene regulating activity of h-TR beta 1.
P19 teratocarcinoma cells differentiate into neurons or muscle when treated with varying doses of retinoic acid, dimethylsulfoxide, thioguanine or butyrate. We induced cardiac differentiation in P19 cells by treating them with 3,5,3'-Triiodo-L-Thyronine (T3). P19 cells received doses of T3 ranging from 30 pM to 300 nM. The beating colonies were counted, and a dose response curve showed that the optimal concentration of T3 was 30 nM. The colonies beat rhythmically for 4-6 weeks, and the cardiac myocytes showed clearly evident cardiac-specific organelles such as nexuses and atrial granules. No evidence of neuronal or skeletal muscle differentiation was seen with any of the concentrations of T3 used. Reverse transcription-polymerase chain reaction showed these cells to express the cardiac ventricular specific marker myosin light chain 2V. T3 is capable of inducing P19 cells to differentiate, in a dose related manner, into spontaneously beating cardiac myocytes identified as such on the basis of ultrastructural criteria. The induction of differentiation is accompanied by expression of cardiac-specific genes. These findings suggest that perhaps genes bearing thyroid response elements in their promoter regions play an important role in the cardiac differentiation induced by T3 in P19 teratocarcinoma cells.
Uptake of the thyroid hormones (TH) 3,5,3'-triiodo-L-thyronine (T3) and L-thyroxine (T4) by trout red blood cells (RBC) was studied by incubating washed RBC in a balanced salts medium containing glucose and [125I]TH at the fish acclimation temperature of 12 degrees. RBC were separated from the medium by centrifugation through silicone oil and glycine buffer (pH 10.5). Maximal [125I]T3 uptake occurred by 10-15 min, but not by 60 min for [125I]T4. First-order uptake was measured at 30 sec for T3 and at 90 sec for T4. Total T4 uptake was enhanced 15-fold from pH 8 to 6 and was affected most below pH 7.2; total T3 uptake was maximal between pH 6.4 and 7.0, but was relatively insensitive to pH. At 0.2 nM, nonsaturable uptake of T3 exceeded that of T4 3- to 6-fold, accounting for 3% (T3) and 50% (T4) of total uptake. Saturable TH uptake was described by Michaelis-Menten kinetics. The saturable transport system for T3 had an apparent K(t) (carrier affinity) of 70-119 nM and J(max) (maximal uptake velocity) of 540-1116 pmol . 10(6) cells(-1) . min(-1). A saturable system was also found for T4, with an apparent K(t) of 99 pM-1.1 nM and J(max) of 8-77 fmol . 10(6) cells(-1) . min(-1). Saturable uptake of both TH depended on temperature. Activation energies for the nonsaturable component were 48 (T4) and 64 (T3) KJ . mol(-1) over the range 0-21 degrees. Activation energies for the saturable components were 52 KJ . mol(-1) (T4, 0-21 degrees), 52 KJ . mol(-1) (T3, 0-10 degrees), and 3 KJ . mol(-1) (T3, 10-21 degrees). During a 16-month study saturable and nonsaturable uptake of both TH increased, probably due to fish age. We conclude that in trout RBC, rapid T3 uptake by a pH- and temperature-sensitive saturable carrier greatly exceeds T4 uptake. The rate of T3 uptake exceeds by 100- to 1000-fold that of mammals and amphibia, and in contrast to those taxa some saturable T4 uptake also occurs.
The effects of short-term food deprivation and photoperiod on plasma thyroid hormone levels of sea bass and sea bream were studied. Animals were acclimated under constant photoperiod regime (15L/9D) and feeding times (2 hr after light onset and 2 hr before light offset). Time-course studies involved monitoring plasma hormone levels every 4 hr throughout 1.5 24-hr cycles. Plasma 3,5, 3'-Triiodo-L-thyronine (T3) and L-thyroxine (T4) were assayed using a newly developed competitive enzyme immunoassay, utilizing acetylcholinesterase as a label of enzymatic tracers. Enzyme immunoassays had sensitivities of 1.25-0.02 and 62.5-0.2 ng/ml for T3 and T4, respectively, and reproducibilities of 3.7 and 5.6% intraassay variation for T3 and T4, respectively; interassay variations for T3 and T4 assays respectively were 1.6%, 11% and 6.6%, 8% for sea bass and sea bream plasma similar to RIA. In sea bass, 3 days of food deprivation resulted in depressed plasma T3 and T4, overriding significant diel variations seen during the second day of starvation. Sea bream displayed a slight decrease of T4 plasma levels while T3 levels remained constant for the whole sampling period. Both thyroidal systems responded to photoperiod with a significant increase in plasma T4 level at the time of light onset. In addition, sea bass also displayed increased T3 levels and decreases in both hormone levels coinciding with "lightoff." Data show different responses of the sea bass and sea bream thyroidal systems to both nutritional state and photoperiod in that the latter state is influenced by the former. Data suggest plasma thyroid levels can be used as a rapid indicator of nutritional status.
Insulin-like growth factor-I (IGF-I) mediates many of the effects of growth hormone (GH). The regulation of IGF-I, independent of GH, is methodologically difficult to assess in vivo, as hypophysectomy results in derangement of many pituitary hormone axes in addition to GH, and a gene knockout model is not available. The recessive sex-linked dwarfing (SLD) gene (dw) in chickens results in a lack of functional target tissue GH receptors due to a variety of molecular defects, which provides a unique model for evaluating GH-independent regulation of IGF-I. In the present study, the impact of 3,3', 5-triiodo-l-thyronine (T3) on circulating and tissue IGF-I was determined in normal versus SLD birds. Adult, nonovulatory female normal and SLD chickens were restrict-fed 40 g of feed/kg bw/day containing 0, 0.5, or 1.0 ppm T3, resulting in supplementation levels of 0 (control), 20 (low dose), or 40 (high dose) microg T3/kg bw/day for 10 days. Samples of GH target tissues including liver, abdominal fat pad, skeletal muscle (pectoralis major), and spleen were extracted and assayed for IGF-I. Plasma T3, T4, GH, and IGF-I were determined by homologous RIA. Tissue GH binding was determined for hepatic membranes by radioreceptor assay. Under control conditions, dwarf chickens were markedly hypersomatotropic (33.3 +/- 4.1 ng GH/ml plasma; mean +/- SEM) compared to normals (2.4 +/- 3.9 ng/ml), and T3 supplementation reduced this to normal levels. Despite the high circulating level of GH in dwarfs, plasma IGF-I was low compared to normal controls (dwarfs 1.5 +/- .9 ng/ml; normals 5. 3 +/- .9 ng/ml; P = 0.004), but this difference was eliminated with low-dose T3. In this study, tissue IGF-I was undetectable in liver and pectoralis muscle in adults (55 weeks of age) of both genotypes, under all treatments. In contrast, adipose tissue IGF-I was relatively high and did not differ (P = 0.84) between genotypes under control conditions (normals 776.5 +/- 236.7; dwarfs 844.6 +/- 236.7 pg/mg protein), but was increased in normals and decreased in dwarfs, resulting in higher levels (P = 0.02) in the normal (1249.9 +/- 200.0 pg/mg protein) than in the dwarf genotype (558.4 +/- 200.0 pg/mg protein) at the higher level of T3 supplementation. This relationship was somewhat reversed in spleen, where T3 tended to decrease tissue IGF-I concentration in normals and increase it in dwarfs. The low level of plasma IGF-I despite nonmeasureable hepatic IGF-I tissue concentrations suggests that IGF-I synthesis by extrahepatic tissues contributes to the circulating pool of IGF-I. The relatively high control levels of adipose tissue IGF-I in the dwarf genotype further suggest that considerable IGF-I synthesis exists that is GH-independent in this extrahepatic tissue. The presence of GH action, however, may mediate the effects of other hormones that can influence local IGF-I production in this tissue, as reflected by the differential response to T3 supplementation between genotypes. The tissue-specific nature of the effect of T3 on IGF-I production supports an additional point of regulation of hormone action at the target tissue level.
An isolated, perfused rainbow trout liver preparation was developed to investigate the action of nonthyroidal hormones on hepatic thyroid hormone metabolism. Several assessments were made of the stability and viability of the preparations under a range of conditions, including measures of lactate dehydrogenase flux and tissue ATP and glycogen content, all of which indicated that the perfused liver was stable for the 60-min perfusion period. Moreover, the liver preparations were responsive to an epinephrine challenge and, throughout the series of experiments, sustained hepatic glucose release. Triiodo-L-thyronine (T3) flux from the liver preparation was significantly increased by the provision of thyroxine (T4) substrate. Epinephrine and bovine thyroid stimulating hormone (TSH) were perfused alone and in combination with T4 to evaluate the effect of these hormones on T3 flux from the liver. Both epinephrine and TSH significantly enhanced hepatic T3 flux in the absence of T3 substrate, but neither had an additional effect on T3 flux when perfused in combination with T4. The results of the study suggest that a relationship exists between the circulating levels of nonthyroid hormones and peripheral thyroid hormone metabolism that may be receptor-mediated.
The effects of thyroid hormone on the production of vitamin A(2) retinoids from vitamin A(1) precursor were investigated in retinal pigmented epithelial (RPE) cells obtained from juvenile coho salmon. Specifically, changes in the production of both the storage and the free forms of radiolabeled 3,4-didehydroretinoids were measured after 24 and 48 h incubation with [11,12-(3)H]all-trans-retinol with (or in the absence of) exogenous 3,4,3'-triiodo-l-thyronine (T(3)). RPE cells incubated with radiolabeled all-trans-retinol showed double the production of radiolabeled 3,4-didehydroretinoids (both storage and free forms) after 48 h compared to 24 h incubation. The addition of T(3) had no significant effect on the production of didehydroretinyl esters (storage form) after 24 h incubation, but did have a significant effect after 48 h, with a ca. 50% reduction in the amount of labeled didehydroretinyl produced compared to the amount produced by the controls after 48 h. Essentially the same observation was made for the production of radiolabeled didehydroretinol (free form) where, after 48 h of incubation with T(3), there was a ca. 50% reduction in the amount of labeled didehydroretinol produced compared to the amount produced by the controls after 48 h. The reduction in both the storage and the free forms of didehydroretinoids indicates that T(3) probably had the effect of altering the activity of the "terminal ring dehydrogenase" enzyme which acts on the outer ring of retinoids. The scope of this experiment, however, does not indicate whether the production of didehydroretinoids is specific to the free or to the storage forms as retinoids within the RPE can shuttle between both forms. The results of this experiment do indicate that the in vitro observations of this experiment may be extended to in vivo and field observations. In vitro, T(3) by itself altered the production of didehydro-derivatives of retinoids. The changes in visual pigment complement seen in salmonids, which are due to changes in relative proportions of 11-cis-retinal and 11-cis-3,4-didehydroretinal in the photoreceptors, may also be the result of T(3) acting by itself on the RPE cells.
The effects of 3,5',3'-triiodo-L-thyronine (T3; 10 or 100 ng ml(-1)), alone or combined with cortisol (500 ng ml(-1)), on the physiological properties of cultured pavement cell epithelia from freshwater rainbow trout gills were assessed. T3 had dose-dependent effects on electrophysiological, biochemical, and ion transporting properties of cultured epithelia in both the absence and the presence of cortisol. These included reduced transepithelial resistance (TER), increased net Na(+) and Cl(-) movement (basolateral to apical) under asymmetrical culture conditions (freshwater apical/L15 media basolateral), and elevated Na(+)-K(+)-ATPase activity. However, paracellular permeability was elevated only in high-dose T3-treated preparations. In T3 + cortisol-treated epithelia, similar T3-induced alterations in TER, net Na(+) and Cl(-) movement, and paracellular permeability were observed, whereas the activity of Na(+)-K(+)-ATPase was further elevated. Under symmetrical culture conditions (L15 medium apical/L15 medium basolateral), T3 had no effect on transepithelial Na(+) and Cl(-) transport, which was passive. However, T3 + cortisol treatment resulted in active Na(+) extrusion (basolateral to apical). Under asymmetrical conditions, hormone treatment did not change the pattern of ion movement (active Na(+) extrusion, active Cl(-) uptake). These experiments demonstrate that cultured pavement cell epithelia from freshwater rainbow trout are T3-responsive and provide evidence for the direct action of T3 and the interaction of T3 and cortisol on the physiology of this preparation.
The effect of 3,3',5-triiodo-L-thyronine (T3) on working memory in ethylcholine aziridinium ion (AF64A)-treated mice was studied in a delayed non-matching to sample task using a T-maze. After behavioural testing was completed, mice were killed by microwave irradiation and regional brain levels of acetylcholine, aspartate, glutamate, glutamine, glycine, taurine, and gamma-aminobutyric acid (GABA) were measured by high-performance liquid chromatography with electrochemical detection. Treatment with AF64A (7 nmol, i.c.v.) produced a deficit in working memory performance in the non-matching to sample task at 30 s delay, and decreased acetylcholine, glutamate, and GABA levels in the hippocampus, but not in the septum and cerebral cortex. Administration of T3 (0.3 mg/kg, p.o., once daily for 6 days) to AF64A-treated animals improved the deficit in working memory performance and reversed the decrease in acetylcholine, glutamate, and GABA levels in the hippocampus. These results indicate that the deficit in performance induced by AF64A can be improved by T3 administration.
The hyperthyroid state is associated with low hepatic glycogen levels, but paradoxically with a high activity of glycogen synthase and low activity of glycogen phosphorylase. We determined the effects of triiodo-L-thyronine (T3) on glycogen synthesis and glycogen synthase activity in rat hepatocytes in vitro. Culture of rat hepatocytes with T3 (100 nM-1 microM) for 16 h-40 h increases glycogen synthesis from glucose and gluconeogenic precursors. The stimulation of glycogen synthesis by T3 was associated with an increase in the activity of glycogen synthase and was additive with the long-term effects of insulin but not with the short-term stimulation of glycogen synthesis by insulin. Culture of hepatocytes with T3 (at concentrations up to 1 microM) did not affect the responsiveness of glycogen synthesis to short-term stimulation by insulin but culture with 10 microM-T3 decreased the responsiveness to insulin without affecting the basal rate. It is suggested that the high activity of glycogen synthase in the hyperthyroid state is due to a direct effect of T3 on the hepatocyte, but the low hepatic glycogen content is probably due to either secondary metabolite and/or endocrine changes or to impaired responsiveness to insulin. T3 may have an anabolic role in the control of hepatic glycogen storage in the euthyroid postprandial state.
We have examined the expression of two nuclear-acting oncogenes, c-fos and c-myc in the rat heart following administration of hormones implicated in the development of cardiac hypertrophy. A single injection of norepinephrine (2.5 micrograms/kg to 2.5 mg/kg) led to transient increases in the levels of both c-fos and c-myc mRNA. The response was sequential: elevated levels of c-fos mRNA were first observed 15 min after treatment and peaked at 1 h whilst c-myc mRNA levels increased 30 min after treatment and peaked at 2 h. The response of both cellular oncogenes to norepinephrine was reduced significantly by alpha blockade but beta blockade was less effective. Administration of triiodo-L-thyronine (0.25 mg/kg), a level known to promote cardiac hypertrophy, did not produce elevated levels of c-fos or c-myc mRNA. In an initial study, it was possible to demonstrate induction of c-fos and c-myc in rat hearts perfused in vitro with medium containing 2 x 10(-7) M norepinephrine. These results provide support for the notion that c-fos and c-myc expression may play a transducing role in the development of adrenergic-mediated, but not thyroid hormone-mediated cardiac hypertrophy.
The effect of triiodo-L-thyronine (T3) treatment (15 mug/100 g body weight daily for 10 days) on the activity of mitochondrial alpha-glycerophosphate dehydrogenase (GPOX) in different nephron segments of the male rat was investigated by a histochemical staining method. The study showed marked segmental differences regarding the response to T3-treatment: 1. The first two proximal segments were unstained in the control rats and intensely stained following treatment. 2. The third proximal segments, the thick ascending limbs of Henle's loop and the distal convolted tubules showed a strong or moderate reaction in controls and a moderate increase after T3-treatment. 3. The high activity of collecting ducts in the cortex and outer zone of the medulla in controls was slightly increased by treatment. 4. Faintly reacting glomeruli and negative thin limbs of Henle's loop and collecting ducts in the inner medulla (papilla) were unaffected by T3-treatment. The results are discussed in relation to biochemical and physiological data.
Recent studies have suggested that acute administration of 3, 5, 3' triiodo-L-thyronine (T3) may increase ventricular performance; however, the direct cellular effects of acute T3 administration are not understood. The objectives of this study were to determine (1) whether T3 acts directly on the cardiac muscle cell (myocyte) itself, and (2) whether T3 acts independently of the myocyte beta-adrenergic receptor. Accordingly, isolated myocyte function was examined using video-microscopy in normal porcine myocytes (n = 60) in the control state and in the presence of increasing T3 concentrations (10 to 500 pmol/L). T3 caused myocyte shortening extent, shortening velocity, and lengthening velocity to increase in a dose-dependent manner. For example, shortening velocity increased from 49.2 +/- 4.3 microns/s at baseline to 66.5 +/- 6.1 microns/s with 100 pmol/L T3 (p < 0.05). beta-Adrenergic stimulation with 25 nmol/L isoproterenol increased shortening velocity to 97.6 +/- 5.7 microns/s; isoproterenol with T3 increased shortening velocity further to 168.5 +/- 10.9 microns/s. Analysis of variance revealed that this increase with T3 was independent of and additive to the beta-adrenergic receptor system. In summary, T3 caused a dose-dependent increase in myocyte contractile performance, and these effects were independent of and additive to beta-adrenergic receptor stimulation. Thus, acute T3 administration may provide a novel modality to improve left ventricular contractile function independent of the beta-adrenergic receptor system.
A spin-labeled derivative of 3,3',5-triiodo-L-thyronine, 3-[( alpha-carboxy-4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenethyl++ +] carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy (SL-T3) has been synthesized. Evaluation of its binding to nuclei after incubation with rat pituitary tumor GH3 cells at 37 degrees C showed that it bound to nuclei with a 18% potency of that of T3. The dynamic interaction of SL-T3 with multilamellar vesicles prepared from dimyristoylphosphatidylcholine (DMPC) was investigated using electron spin resonance techniques. At 31 degrees C, the lateral diffusion constant of SL-T3 in DMPC membranes was found to be 3.0 X 10(-8) cm2/s as determined by the ESR line-broadening method. The temperature dependency of the ESR spectrum of SL-T3 in DMPC multilamellar vesicles showed a break at 23.5 degrees C, which is close to the main phase-transition temperature, 23.7 degrees C, of DMPC membranes. This suggests that the motion of the probe reflects the motion of phospholipids in DMPC membranes, and that the probe itself does not perturb the membrane structure. SL-T3 appears to be a useful probe for studying the motion of thyroid hormone in the plasma membrane of responsive cells.
To develop antibodies against the plasma membrane 3,3',5-triiodo-L-thyronine (T3) binding protein (M.W. 55,000), rabbits were immunized with formalin-fixed GH3 cells or highly purified plasma membranes from these cells. Antibodies were screened by immunoprecipitation using detergent solubilized N-bromoacetyl-[125I]T3-labeled 55K protein. Among the nine detergents tested, 0.18% CHAPS was found to be the best in its solubilization efficiency and its ability to maintain the integrity of the antigenicity of the 55K protein. The N-bromoacetyl-[125I]T3-labeled 55K protein was also immunoprecipitated by anti-T3 antibodies. The anti-55K protein antibodies cross-reacted with plasma membrane T3 binding proteins from cultured cells and tissues of human and rodent origin. These results indicate that structural similarities exist in human and rodent plasma membrane T3 binding proteins. These antibodies should provide a powerful tool in the characterization and in probing the function(s) of the plasma membrane T3 binding protein in cells.