The crystal structure of a major metabolite of thyroid hormone: 3,3′,5′-triiodo-L-thyronine

Two independent conformations of the thyroinactive thyroid hormone metabolite, 3,3′,5′-triido-L-thyronine (rT₃) were determined by X-ray diffraction methods. The conformations show significant difference in the lettering geometry when compared with those of the thyroactive thyroxine (T₄) and 3,5,3′-triido-L-thyronine (T₃). The diphenyl ether conformation of the two conformers of rT₃ is an anti-skewed one, in which the torsion angels, φ (C5-C4-O4-Cl′) are 8° and ?6°, and φ′ are 86° and 87°. This conformation is in contrast to a twist-skewed one of T₄ and T₃. The difference in the binding abilities between T₄, T₃ and rT₃ to thyroxine binding carrier proteins in serum or to a nuclear receptor protein may be explained by the characteristics solid-state conformations of these metabolites.     

Circulating reverse triiodothyronine (rT3) in the dog

Plasma reverse triiodothyronine (rT₃) concentration was measured by radio immunoassay (RIA) in a group of 15 dogs. The mean rT₃ concentration was 187 ng/100 ml which was 3 times higher than radioimmunoassayable triiodothyronine (T₃) concentration. rT₃ measurement in thyroid and peripheral venous plasma in 3 dogs showed that the unusually high circulating rT₃ levels in this species could not be explained on the basis of augmented thyroidal rT₃ secretion. Study of rT₃-protein binding by equilibrium dialysis also failed to show any evidence of unusual rT₃-protein interaction (rT₃ free fraction was 2 — 3 times greater than in normal human serum). Among all the species examined so far (man, monkey, sheep, dog and rat), only in the dog are the circulating rT₃ levels significantly higher than T₃ suggesting that in this species the 5-deiodination, in marked contrast to the 5'-deiodination noted in several other species, is a major pathway normally involved in the initial monodeiodination of T₄.     

Neurobiology and pharmacology of Huntington's disease.

Reverse triiodothyronine (rT₃) which is apparently a product of 5-monodeiodination of thyroxine (T₄) at the periphery, was measured in 199 chickens of various strains. rT₃ was virtually absent in young birds (less than 1 week to 8 weeks of age) in marked contrast to the elevated rT₃ levels found in human and other mammalian neonates. At one to two years of age there was a significant increase in the number of birds with detectable rT₃. However, rT₃ concentrations were low and often close to the detection limits of the assay in contrast to significant rT₃ levels found in mammals (man, monkey, sheep, rat and dog). An apparent sex difference in relation to rT₃ formation was noted; 46.4% of 97 females and 9.3% of 54 males showed detectable rT₃ levels. The observations described suggest a species difference in regard to peripheral T₄ monodeiodination between birds and mammals.     

Different pathways of iodothyronine 5′-deiodination in rat cerebral cortex

Microsomal fractions of rat cerebral cortex catalyze the 5′-deiodination of 3,3′,5′-triiodothyronine (rT₃) in the presence of thiols such as dithiothreitol. Evidence is presented that two different enzymatic pathways are involved. One of these has a low apparent K_m (2.7 nM) for rT₃, is inhibited by nanomolar concentrations of thyroxine (T₄), but not by up to 1 mM 6-propyl-2-thiouracil (PTU). The other pathway has a high apparent K_m (31 nM) for rT₃, is inhibited by PTU, but not by <1 μM T₄. The relative proportion of rT₃ 5′-deiodination via either pathway depends on thyroid status, with increased contributions from the low-K_m system especially in short-term hypothyroidism.     

Serum principal iodothyronines and the influence of cold exposure associated with shearing in the sheep

The effect of cold exposure caused by shearing on serum thyroid hormone (TH) concentrations in sheep kept at an ambient temperature of 8.5°C was studied. While the deep body temperature fell to the lowest level 4 h after shearing the concentration of triiodothyronine (T₃) increased to a peak value at that time. Thyroxine (T₄) and metabolically inactive reverse triiodothyronine (rT₃) levels reached their peak value after 24 h. The $\text{T}{}_3\text{T}{}_4$ ratio reached a maximum at about 4 h and $\text{rT}{}_3\text{T}{}_4$ and $\text{rT}{}_3\text{T}{}_3$ ratios rose to maximum values about 24 h after shearing. This sequence of events suggest a biphasic response to cold—an immediate secretion of TH from the thyroid gland, followed by adaptive alteration in T₃ and rT₃ generation in the extrathyroidal tissues.     

The effect of propylthiouracil on thyroid-stimulating hormone-induced alterations in iodothyronine secretion from perfused dog thyroids

The aim of this study was to see whether the inhibitory effect of propylthiouracil on thyroidal secretion of 3,5,3′-triiodothyronine (T₃) and 3,3′,5′-triiodothyronine (rT₃) could be reproduced in intensively stimulated thyroids, and to elucidate whether an increase in the fractional deiodination of thyroxine (T₄) to T₃ and rT₃ during iodothyronine secretion might be responsible for the transient fall in the T₄/T₃ and T₄/rT₃ ratios in thyroid secretion seen in the early phase after stimulation of thyroid secretion.For this purpose T₄, T₃ and rT₃ were measured in effluent from isolated dog thyroid lobes perfused in a non-recirculation system using a synthetic hormone free medium. 1 mmol/l propylthiouracil induced a significant reduction in thyroid-stimulating hormone (TSH) stimulated T₃ and rT₃ release while the release of T₄ was unaffected. This supports our previous conclusion that T₄ is partially monodeiodinated to T₃ and rT₃ during thyroid secretion. Infusion of 1 mmol/l propylthiouracil for 30 min or 3 mmol/l propylthiouracil for 120 min did not abolish the transient fall in effluent T₄/T₃ and T₄/rT₃ induced by TSH stimulation. Thus, this phenomenon seems not to depend on intrathyroidal iodothyromine deiodinating processes.     

Phenolic and nonphenolic ring deiodinations of iodothyronines in cultured hepatocarcinoma cell hemogenate from monkey

Iodothyronine monodeiodinase activities in homogenates of cultured monkey hepatocarcinoma cells were measured by the deiodination of [3,5-¹²⁵I]triido-l-thyronine or 3-[3′5′-¹²⁵I]triido-l-thyronine (phenolic ring-labeled ‘reverse’ triiodothyronine). The assay system utilized a small ion-exchange column (AG50W-X4, 0.9×～1 cm) to measure ¹²⁵I^?. Both deiodinases were destroyed by boiling for 1 min.Maximal nonphenolic ring deiodination was observed at pH 7.9 whereas maximal phenolic ring deiodination was at pH 6.3. Both reactions were enhanced strongly by dithiothreitol (0.1–5 mM), and slightly by 5 mM β-mercaptoethanol. Phenolic ring deiodination was strongly inhibited by 0.1 mM propylthiouracil. Nonphenolic ring deiodination was accelerated by EDTA (1.2 mM) and inhibited by Mg²⁺ (5 mM). Methylmercaptoimidazol and Mg²⁺, Ca²⁺ and Mn²⁺ (0.1–1.0 mM) had little or no effect on either reaction, but Zn²⁺ (0.1 mM) strongly inhibited both.Both reactions were inhibited by excess iodothyronine analogues at 10 mM to 10μM, and thyroxine was shown to be a competitive inhibitor in both cases. On the basis of relative affinities and inhibitory effects, it appears that the order of affinity for the phenolic ring deiodinase is 3,3′,5′-triiodo-l-thyronine-(rT₃) > l-thyroxine(T₄) > 3,4,3′-triido-l-thyronine(T₃), whereas for the nonphenolic ring deiodinase the order is T₃ > T₄ > rT₃. Diiodotyrosine did not affect their deiodination.     

Effects of thyromimetric drugs on aldosterone-dependent sodium transport in the toad bladder

Summary Aldosterone increases transepithelial Na⁺ transport in the urinary bladder ofBufo marinus. The response is characterized by 3 distinct phases: 1) a lag period of about 60 min, ii) an initial phase (early response) of about 2 hr during which Na⁺ transport increases rapidly and transepithelial electrical resistance falls, and iii) a late phase (late response) of about 4 to 6 hr during which Na⁺ transport still increases significantly but with very little change in resistance. Triiodothyronine (T₃, 6nm) added either 2 or 18 hr before aldosterone selectively antagonizes the late response. T₃ per se (up to 6nm) has no effect on base-line Na⁺ transport. The antagonist activity of T₃ is only apparent after a latent period of about 6 to 8 hr. It is not rapidly reversible after a 4-hr washout of the hormone. The effects appear to be selective for thyromimetic drugs since reverse T₃ (rT₃) is inactive and isopropyldiiodothyronine (isoT₂) is more active than T₃. The relative activity of these analogs corresponds to their relative affinity for T₃ nuclear binding sites which we have previously described. Our data suggest that T₃ might control the expression of aldosterone by regulating gene expression, e.g. by the induction of specific proteins, which in turn will inhibit the late mineralocorticoid response, without interaction with the early response.     

Effects of ovine prolactin on iodide metabolism and thyroid activity in the eel

Summary In the eel, ovine prolactin (oPrl) treatment (0.018 IU/day·g body weight), for 8 to 13 days modifies neither iodide absorption from the water nor excretion, extrathyroidal metabolism and plasma level of iodide.Thyroid activity, evaluated by epithelial cell height, radioiodine uptake and absolute iodide uptake is approximately twice that of controls. However, the amounts of total iodine, thyroxine (T₄) and triiodothyronine (T₃) in thyroid are unaltered by oPrl. Therefore, the decrease of plasma T₄ and the increase of plasma T₃, previously observed in oPrl-treated eels, do not result from a preferential thyroidal secretion of T₃, but only from a stimulation of peripheral conversion of T₄ to T₃. Furthermore, the increased thyroid activity probably originates from a decreased feedback inhibition following the fall of circulating T₄ induced by oPrl.Abbreviations oPrl ovine prolactin - T ₄ Thyroxine - T ₃ 3.5.3 triiodothyronine - TRH thyrotropin releasing hormone - TSH thyroid stimulating hormone - PBI protein bound iodine     

A method for the analysis of six thyroid hormones in thyroid gland by liquid chromatography–tandem mass spectrometry

Perchlorate can competitively inhibit iodide uptake by the thyroid gland (TG) via the sodium/iodide symporter, consequently reducing the production of thyroid hormones (THs). Until recently, the effects of perchlorate on TH homeostasis are being examined through measurement of serum levels of TH, by immunoassay (IA)-based methods. IA methods are fast, but for TH analysis, they are compromised by the lack of adequate specificity. Therefore, selective and sensitive methods for the analysis of THs in TG are needed, for assessment of the effects of perchlorate on TH homeostasis. In this study, we developed a method for the analysis of six THs: l-thyroxine (T₄), 3,3′,5-triiodo-l-thyronine (T₃), 3,3′,5′-triiodo-l-thyronine (rT₃), 3,5-diiodo-l-thyronine (3,5-T₂), 3,3′-diiodo-l-thyronine (3,3′-T₂), and 3-iodo-l-thyronine (3-T₁) in TG, using liquid chromatography (LC)–tandem mass spectrometry (MS/MS). TGs used in this study were from rats that had been placed on either iodide-deficient diet or iodide-sufficient diet, and that had either been provided with perchlorate in drinking water (10 mg/kg/day) or control water. TGs were extracted by pronase digestion and then analyzed by LC–MS/MS. The instrumental calibration range for each TH ranged from 1 to 200 ng/ml and showed a high linearity (r > 0.99). The method quantification limits (LOQs) were determined to be 0.25 ng/mg TG for 3-T₁; 0.33 ng/mg TG for 3,3′- and 3,5-T₂; and 0.52 ng/mg TG for rT₃, T₃, and T₄. Rats were placed on an iodide-deficient or -sufficient diet for 2.5 months, and for the last 2 weeks of that period were provided either perchlorate (10 mg/kg/day) in drinking water or control water. Iodide deficiency and perchlorate administration both reduced TG stores of rT₃, T₃, and T₄. In iodide-deficient rats, perchlorate exacerbated the reduction in levels of THs in TG. With the advances in analytical methodology, the use of LC–MS/MS for measurement of hormone levels in TG will allow more comprehensive evaluations of the hypothalamic-pituitary–thyroid axis.     

Phenolic ring deiodination in cultured rat hepatoma cells, and subcellular localization of deiodinases in cultured rat hepatoma, monkey hepatocarcinoma cells and normal rat liver homogenates

The cultured rat hepatoma cell (R117-21B) homogenates metabolized 3,[3′,5′-¹²⁵I]triiodothyronine by phenolic ring deiodination and produced radioactive iodide and 3,3′-diiodothyronine. Thyroxine (T₄) was converted to 3,3′,5-triidothyronine (T₃). The production of ¹²⁵I⁻ presented the deiodinase activity. The optimal pH for phenolic ring deiodination was observed to be pH 6.0–7.0. This enzyme reaction was accelerated by dithiothreitol. Propylthiouracil strongly inhibited the phenolic ring deiodination at 0.1 mM, whereas an effect of 20 mM methylmercaptoimidazol on the deiodination was very weak or absent.Excess unlabeled iodothyronines (T₄, T₃ and 3,5-diiodo-l-thyronine inhibited the phenolic ring deiodination of labeled 3,3′,5′-triiodothyronine, althought their inhibitory effect was slightly different. Triiodothyroacetic acid was a better inhibitor than T₃. Diiodotyrosine did not affect phenolic ring deiodination in cultured rat hepatoma cell homogenates.Phenolic and nonphenolic ring deiodinase activities of cultured monkey hepatocarcinoma cell and rat liver homogenates were also studied by the use of 3,[3′,5′-¹²⁵I]triiodothyronine and [3,5-¹²⁵I]thyroxine, respectively. Both deiodinase activities were observed in particulate fractions (mitochondrial and microsomal) of cultured cell and rat liver homogenates.     

Regulation of thyroid hormone metabolism in rat liver fractions.

The nature of the conversion of thyroxine (T4) to triiodothyronine (T3) and reverse triiodothyronine (rT3) was investigated in rat liver homogenate and microsomes. A 6-fold rise of T3 and 2.5-fold rise of rT3 levels determined by specific radioimmunoassays was observed over 6 h after the addition of T4. An enzymic process is suggested that converts T4 to T3 and rT3. For T3 the optimal pH is 6 and for rT3, 9.5. The converting activity for both T3 and rT3 is temperature dependent and can be suppressed by heat, H2O2, merthiolate and by 5-propyl-2-thiouracil. rT3 and to a lesser degree iodide, were able to inhibit the production of T3 in a dose related fashion. Therefore the pH dependency, rT3 and iodide may regulate the availability of T3 or rT3 depending on the metabolic requirements of thyroid hormones.     

Triiodothyronine but Not Thyroxine Accelerates Myofibrillar Proteolysis via ATP Production in Cultured Muscle Cells

These experiments were done to clarify that the differential effects of thyroxine (T₄) and triiodothyronine (T₃) on skeletal muscle protein turnover are caused by their roles on ATP production. Primary cultured chick muscle cells were treated with a physiological level of T₄ (60 ng/ml), T₃ (12 ng/ml), or ATP (0.5 mM) for 6 days and the protein content, ATP production, proteasome activity, and myofibrillar protein breakdown were measured. The protein content measured as an index of cell growth was not affected by T₄, T₃, or ATP. The cellular ATP level was increased by T₃ and ATP, but not by T₄. Proteasome activity and N ^τ-methylhistidine (MeHis) release measured as an index of myofiblillar protein breakdown was also increased by T₃ and ATP, but not by T₄. These results indicate that T₃ but not T₄ increases ATP production followed by an increase in proteasome activity, and thus stimulates myofibrillar proteolysis.     

New ionic targets of 3,3′,5′-triiodothyronine at the plasma membrane of rat Sertoli cells

The functions of Sertoli cells, which structurally and functionally support ongoing spermatogenesis, are effectively modulated by thyroid hormones, amongst other molecules. We investigated the mechanism of action of rT₃ on calcium (⁴⁵Ca²⁺) uptake in Sertoli cells by means of in vitro acute incubation. In addition, we performed electrophysiological recordings of potassium efflux in order to understand the cell repolarization, coupled to the calcium uptake triggered by rT₃. Our results indicate that rT₃ induces nongenomic responses, as a rapid activation of whole-cell potassium currents in response to rT₃ occurred in <5 min in Sertoli cells. In addition, the rT₃ metabolite, T₂, also exerted a rapid effect on calcium uptake in immature rat testis and in Sertoli cells. rT₃ also modulated calcium uptake, which occurred within seconds via the action of selective ionic channels and the Na⁺/K⁺ ATPase pump. The rapid response of rT₃ is essentially triggered by calcium uptake and cell repolarization, which appear to mediate the secretory functions of Sertoli cells.     

Rapid Responses to Reverse T3 Hormone in Immature Rat Sertoli Cells: Calcium Uptake and Exocytosis Mediated by Integrin

There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T₃ (rT₃) action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT₃ are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT₃ was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT₃-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions). Therefore, our findings suggest that calcium uptake stimulated by rT₃ may be mediated by integrin α_vβ₃. In addition, it was demonstrated that calcium uptake stimulated by rT₃ is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT₃ also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT₃ modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology.     

Separate mechanisms of induction for ornithine decarboxylase by triiodothyronine and aminophylline

A single dose of aminophylline (200 μmol/kg, i.p.) or triiodothyronine (T₃, 300 μg/kg, i.p.) resulted in the induction of ornithine decarboxylase (ODC) in rat liver with maximal activity 10-fold and 6-fold above controls, respectively, 4 hr after the administration of the drug or hormone. After either agent, the induction of ODC was blocked by either cycloheximide or actinomycin D. The same concentrations of aminophylline and T₃ administered simultaneously produced an additive 16-fold increase in ODC activity. After T₃ administration, the cyclic AMP-dependent protein kinase activity ratio was unaltered at all times measured. After aminophylline, the protein kinase activity ratio was elevated by 15 min and remained elevated for 2 hr. Somatostatin administration (50 μg/100 g), which lowers plasma growth hormone to 30% of control, had no effect on the ability of T₃ to induce ODC. These data suggest separate routes of induction of ODC in response to aminophylline and T₃. Aminophylline induction occurs via cycyclic AMP-mediated event whereas T₃ does not involve ccyclic AMP but results from a direct nuclear interaction.     

The rapid transient stimulation of both cytoplasmic and mitochondrial ornithine decarboxylases in the liver of thyroidectomized rats by 3,5,3′-triiodo-L-thyronine

Evidence is presented that liver from thyroidectomized rats has ornithine decarboxylase (ODC) in both mitochondrial and soluble fractions. The cytosolic activity was stimulated 7-fold and the mitochondrial activity 3-fold 15 min after the administration of triiodothyronine (T₃) . While the rapid transient stimulation of ODC could represent a direct intracellular response to T₃, an incidental effect on a very-rapidly-turning-over enzyme seems more likely; this suggests thatin vivo ODC may be controlled by a demand for polyamines.     

Measurement and regulation of thyroidal status in teleost fish

Summary We have reviewed the stages in teleost thyroid function and its regulation, from the initial biosynthesis of the TH to their eventual interaction with putative receptors.TH biosynthesis depends on an adequate plasma iodide level, determined partly by dietary iodide and partly by active branchial iodide uptake from the water, Pulse-injected radioiodide can be used to evaluate thyroidal iodide uptake, aspects of TH biosynthesis and TH thyroidal secretion. However, owing to variable plasma iodide levels, care is required in interpretating these parameters. TH biosynthesis, thyroglobulin properties and intrathyroidal secretion mechanisms have received limited recent attention. Histological indices of thyroid tissue changes, while useful in many situations, do not always correlate with more direct estimates of thyroidal secretion and can be misleading.Thyroid function is regulated by the hypothalamo-pituitary-thyroid axis, but neither the identities of the hypothalamic factors nor a reliable immunoassay for TSH have been established. Currently, activity of the hypothalamic-pituitary axis is usually determined by pituitary thyrotrope histological appearance or bioassay of pituitary TSH. Plasma free T₄ feeds back at both the pituitary and hypothalamic levels and inhibits TSH release. Thyroidal T₄ secretory activity is presumably adjusted to maintain a constant plasma T₄level according to physiologic state.Plasma T₄ is probably the most commonly used index of thyroidal status. However, (1) T₄ is probably not the active form of TH, (2) the T₄ plasma level may be influenced by the binding properties of plasma proteins, and (3) the T₄ concentration alone makes no provision for the rate of T₄ turnover in plasma. The most practical way to measure thyroidal T₄SR is to determine plasma T₄DR, and assuming steady-state conditions, equate it to T₄SR. The T₄DR is determined from kinetic studies employing^*T₄, which also enable estimates of sizes of vascular and extravascular T₄ pools and their rates of exchange. Excretion of T₄ or its derivatives in urine or bile can be determined also. A high proportion of T₄ is enzymatically monodeiodinated in liver and other tissues, generating T₃ for local (intracellular) and vascular systemic compartments.Bothin vivo andin vitro methods have been used to quantify T₄ deiodinase activity, which is highly responsive to physiologic state and environmental variables. T₃ production is inhibited by a moderate T₃ excess indicating an autoregulatory system, whereby tissue T₃ levels are maintained at a set-point appropriate for a particular physiologic state. The rate of T₃ production provides an informative measure of thyroidal status in a given tissue. However, other pathways also contribute to the maintenance of T₃ homeostasis at a particular set-point. These include the rate of T₃ degradation to 3,3-T₂, the rate of T₄ substrate diversion to rT₃ (an inactive isomer) and by the excretion of parent compounds or conjugates in bile and urine. Potential losses across branchial or integumentary surfaces have yet to be evaluated.The most fundamental measure of thyroidal status is represented by the amount of T₃ saturably bound to receptors/nucleus for the cell type of interest. This is estimated most accurately in double isotope studies in which T₃ contributions from both vascular and intracellular compartments are evaluated. Less satisfactory but meaningful indices of T₃ availability to receptor sites may be obtained from the plasma T₃ (or free T₃) level and from the tissue T₃ level. The former is appropriate if the cell type in question obtains its T₃ primarily from plasma; the latter should be measured if the cell type derives its T₃ mainly through intracellular deiodinase activity. If the proportion of vascular T₃/intracellular T₃ bound to receptors is known, it may indicate the degree of receptor activation. However, even cytosolic T₃ levels may not vary in proportion to nuclear T₃ levels.Differences in thyroidal function between teleosts and homeotherms can be attributed to distinctive strategies in iodide economy and to fundamental differences in control of thyroidal status. Owing to more certain iodide availability (branchial iodide pump and plasma iodide-binding proteins), teleosts are probably more liberal in their iodide use and have less efficient mechanisms for recovery and retention of hormonal iodide than homeotherms. Also, primary control of teleost thyroidal function appears peripheral. It is the finely regulated conversion of T₄ to T₃ in tissues which may largely determine the T₄ secretion rate. Thus, T₄, as a prohormone, may be produced more to satisfy the substrate needs for T₄ conversion rather than to drive T₃ production. Because TH are mainly implicated in tissue- or cell-specific processes involved in development, growth and reproduction in teleosts, it may be advantageous for their thyroidal status to be determined locally through T₄-to-T₃ deiodination. In homeotherms, primary control is mainly central through the hypothalamic-pituitary axis, which regulates thyroidal secretion of T₄ and significant amounts of T₃. The level of T₄ (free T₄) is believed to drive the production of T₃ in most peripheral tissues. Because TH are extensively involved in the systemically integrated adjustment of basal metabolic rate in homeotherms, it may have been advantageous to evolve a system leaning towards central control by the hypothalamus, the brain centre associated with thermoregulation.     

Effect of insulin-induced hypoglycemia on the serum concentrations of thyroxine,triiodothyronine and reverse triiodothyronine

The effect of insulin-induced hypoglycemia on serum thyroid hormone concentrations was studied in nine healthy individuals. Before, during and after the hypoglycemia blood samples were taken for measurement of the concentrations of glucose, thyroxine (T₄), triiodothyronine (T₃), reverse triiodothyronine (rT₃), catecholamines and pituitary hormones.There was no change in the mean serum T₄ level (± the standard error of the mean) of 67 ± 2 μg/l. However, the T₃ concentrations rose from a mean basal level of 1.86 ± 0.06 μg/l to a mean peak of 2.51 ± 0.21 μg/l (P < 0.01) at 45 minutes after the insulin injection, and the rT₃ concentrations fell from a mean basal level of 0.184 ± 0.008 μg/l to a mean nadir of 0.171 ± 0.022 μg/l (not a significant change). The mean peak epinephrine level was 545 ± 103 ng/l and it occurred between 30 and 45 minutes after the insulin injection; the mean peak norepinephrine level was 584 ± 114 ng/l and it occurred between 30 and 90 minutes after the injection. The growth hormone levels reached a mean peak of 26.1 ± 4.8 μg/l and the plasma cortisol levels rose to 215 ± 9 μg/l. The mean basal prolactin level was 8.5 ± 0.9 μg/l; in five subjects there was a rise to a mean peak of 50.6 ± 14.6 μg/l, whereas in the remaining four no significant increase occurred. No correlation was found between the changes in the serum T₃ concentration and any of the other factors studied.It was concluded that acute hypoglycemia is associated with a rapid increase in the serum T₃ concentration.