T4 Release Research Articles

Changes in glucose, glycogen, thyroid activity and hypothalamic catecholamines in tench by starvation and refeeding.

The effects of short-term food deprivation (7 days) and refeeding (2 days) on different biochemical and neuroendocrine parameters were studied in tench. A 7-days fast resulted in a significant reduction of plasma glucose and glycogen hepatic content, supporting the key role of liver glycogen as energy depot for being consumed during fasting. The rapid recovery of normal values of blood glucose and glycogen stores by refeeding indicates a rapid replenishment of liver glycogen stores. The short-term starvation decreased circulating thyroid hormones (both T3 and T4) and T4 release from thyroid, supporting an interaction between nutritional state and thyroid function in tench. All these metabolic and hormonal changes were partial or totally reversed under refeeding conditions. An increase in hypothalamic content of norepinephrine and dopamine was found in fasted fish. This result might be a consequence of stress induced by starvation.

Targeting of thyroglobulin to transcytosis following megalin-mediated endocytosis: evidence for a preferential pH-independent pathway.

TG internalized from the colloid by megalin, bypasses the lysosomal pathway and is transported across thyrocytes by transcytosis. Although most of the intracellular mechanisms responsible for targeting of ligands to transcytosis are unknown, for certain ligands a role of lysosomal pH has been established. Thus, ligands that undergo lysosomal degradation dissociate from their receptors due to the low pH of endosomes, whereas certain ligands that undergo transcytosis fail to dissociate because they bind to their receptors at acidic pH. Here we studied the role of pH in TG transcytosis. We first investigated the effect of pH on megalin binding to TG in solid phase assays and found that, although megalin bound to TG at various pH values (ranging from 4-8), optimal binding was seen at acidic pH (ranging from 4.5-6). We then studied the effect of chloroquine (CQ) and ammonium chloride (AC), which increase endosomal pH, on transcytosis of TG across Fisher rat thyroid (FRTL-5 cells). Transcytosis assays were performed using FRTL-5 cells cultured on filters in dual chambered devices, with megalin expression only on the upper surface of the layers. TG was added to the upper chamber and transcytosed TG was measured in fluids collected from the lower chamber after incubation at 37 C. Treatment of FRTL-5 cells with CQ or AC did not affect binding and uptake of TG, but it did reduce T3 release from exogenously added TG, used as a measure of TG degradation in the lysosomal pathway. Treatment with CQ or AC resulted in an increase of transcytosis of TG across FRTL-5 cells, but only to a minimal extent (15-20%). The effects of CQ or AC and those of a megalin competitor (the monoclonal antibody 1H2, which reduced transcytosis) were not additive, suggesting that CQ and AC act on the megalin-mediated pathway. In conclusion, because TG binding to megalin is greatest at acidic pH, it is possible that TG does not dissociate from megalin in the lysosomal pathway. However, the pH-dependence of TG binding to megalin does not account for much of transcytosis, which probably occurs largely because of other mechanisms of targeting.

Seasonal changes of thyroid hormones in field-collected Atlantic cod in relation to condition indices, water temperature and photoperiod

Serum T4 and T 3 in wild Atlantic cod Gadus morhua ranged from 1 to 12 ng ml - and from 2 to 27 ng ml -1 respectively over a 3-year period. In general, the concentrations increased from summer (T 3 ) or early autumn (T 4 ) to maxima in mid-winter and declined abruptly during spring. The T 4 /T 3 monthly means were lowest in summer and highest in winter. The seasonal patterns of thyroid hormones were weakly correlated with changes in water temperature. However, both T4 and T 3 co-varied simultaneously with photoperiod. In addition, T 3 was correlated with the hepatosomatic index and condition factor during summer and autumn. It is suggested that the seasonal changes in the release of T4 from the thyroid were photoperioddriven, and that the course of T 3 was regulated by the metabolic state of the fish during the somatic growth period.

Leptin, nutrition, and the thyroid: the why, the wherefore, and the wiring

The function of the thyroid gland is to produce the thyroid hormones T3 and T4, which regulate gene transcription throughout the body (1). In medical practice, the thyroid becomes an issue when its size or shape becomes abnormal or when it produces too much or too little hormone. Thus, we typically think of the thyroid with reference to the clinical states of goiter, or hyper- or hypothyroidism. But what is the physiology of the thyroid when the gland and the entire hypothalamic-pituitary-thyroid axis are intact? As first year medical students ask each year, Why exactly do we have a thyroid, at all?

Role of Megalin (gp330) in Transcytosis of Thyroglobulin by Thyroid Cells: A NOVEL FUNCTION IN THE CONTROL OF THYROID HORMONE RELEASE

When thyroglobulin (Tg) is endocytosed by thyrocytes and transported to lysosomes, thyroid hormones (T4 and T3) are released. However, some internalized Tg is transcytosed intact into the bloodstream, thereby avoiding proteolytic cleavage. Here we show that megalin (gp330), a Tg receptor on thyroid cells, plays a role in Tg transcytosis. Following incubation with exogenous rat Tg at 37 degrees C, Fisher rat thyroid (FRTL-5) cells, a differentiated thyroid cell line, released T3 into the medium. However, when cells were incubated with Tg plus either of two megalin competitors, T3 release was increased, suggesting that Tg internalized by megalin bypassed the lysosomal pathway, possibly with release of undegraded Tg from cells. To assess this possibility, we performed experiments in which FRTL-5 cells were incubated with either unlabeled or (125)I-labeled Tg at 37 degrees C to allow internalization, treated with heparin to remove cell surface-bound Tg, and further incubated at 37 degrees C to allow Tg release. Intact 330-kDa Tg was released into the medium, and the amount released was markedly reduced by megalin competitors. To investigate whether Tg release resulted from transcytosis, we studied FRTL-5 cells cultured as polarized layers with tight junctions on permeable filters in the upper chamber of dual chambered devices. Following the addition of Tg to the upper chamber and incubation at 37 degrees C, intact 330-kDa Tg was found in fluids collected from the lower chamber. The amount recovered was markedly reduced by megalin competitors, indicating that megalin mediates Tg transcytosis. We also studied Tg transcytosis in vivo, using a rat model of goiter induced by aminotriazole, in which increased release of thyrotropin induces massive colloid endocytosis. This was associated with increased megalin expression on thyrocytes and increased serum Tg levels, with reduced serum T3 levels, supporting the conclusion that megalin mediates Tg transcytosis. Tg transcytosis is a novel function of megalin, which usually transports ligands to lysosomes. Megalin-mediated transcytosis may regulate the extent of thyroid hormone release.

The thyroxine-binding proteins.

The slow clearance, prolonged half-life, and high serum concentration of thyroxine (T4) are largely due to strong binding by the principal plasma thyroid hormone-binding proteins, thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin. These proteins, which shield the hydrophobic thyroid hormones from their aqueous environment, buffer a stable free T4 concentration for cell uptake. Free rather than bound T4 is subject to homeostatic control by the hypothalamic-pituitary thyroid axis. Although it is not a protease inhibitor, sequence analysis identifies TBG as a member of the serine protease inhibitor (serpin) family of proteins. Proteolytic cleavage of TBG appears to be a mechanism for site-specific release of T4 independently of homeostatic control. TBG probably facilitates the transport of maternal T4 and iodide to the fetus, although this remains to be proven. High-affinity cellular binding sites for TTR have been described; however, their function and that of choroid plexus synthesis of TTR and transport of T4 into the cerebrospinal fluid remain unclear. Albumin, with the lowest T4 affinity and fastest T4 release of the major T4-binding proteins may promote quick exchange of T4 with tissue sites. The affinity of albumin for T4 is increased by histidine substitution for arginine 218 in the most common form of dysalbuminemic hyperthyroxinemia. However, proline and alanine substitutions at the same site have a similar effect, suggesting that arginine 218 interferes with T4 binding.

Type 2 iodothyronine deiodinase expression in the cochlea before the onset of hearing.

Thyroid hormone signaling during a postnatal period in the mouse is essential for cochlear development and the subsequent onset of hearing. To study the control of this temporal dependency, we investigated the role of iodothyronine deiodinases, which in target tissues convert the prohormone thyroxine into triiodothyronine (T3), the active ligand for the thyroid hormone receptor (TR). Type 2 5'-deiodinase (D2) activity rose dramatically in the mouse cochlea to peak around postnatal day 7 (P7), after which activity declined by P10. This activity peak a few days before the onset of hearing suggests a role for D2 in amplifying local T3 levels at a critical stage of cochlear development. A mouse cochlear D2 cDNA was isolated and demonstrated near identity to rat D2. In situ hybridization localized D2 mRNA in periosteal connective tissue in the modiolus, the cochlear outer capsule and the septal divisions between the turns of the cochlea. Surprisingly, D2 expression in these regions that give rise to the bony labyrinth was complementary to TR expression in the sensory epithelium. Thus, the connective tissue may control deiodination of thyroxine and release of T3 to confer a paracrine-like control of TR activation. These results suggest that temporal and spatial control of ligand availability conferred by D2 provides an unexpectedly important level of regulation of the TR pathways required for cochlear maturation.

Ontogeny of thyroid tissue and tissue thyroid hormone clearance in rainbow trout embryos reared at two temperatures

The development of the thyroid tissue and clearance of thyroid hormones was studied in rainbow trout embryos reared at 8.5 and 5.5 °C, in order to determine if these events are affected by temperature in the same manner as growth rate. Although the change in growth rate was found to be predictable using the degree-day model, the pattern of whole body thyroid hormone clearance was not similarly related to rearing temperature. Further, although the thyroid follicles appeared at different times in embryos reared at the two temperatures, the differences could not be predicted using the degree-day model. These results suggest that the development of the thyroid tissue and the clearance of yolk hormones are not directly related to rearing temperature. Immunohistochemical staining of the thyroid tissue indicated two phases of thyroid activity, one associated with hormonogenesis, and the other a later phase of hormone release. However, there was no increase in 'whole body' thyroid hormone level associated with the time of appearance of colloid in the follicles. Further, evidence was presented to suggest that the extraction method used did not remove thyroid hormone associated with the colloid in the follicles. There was also evidence of sporadic increases in 'whole body' thyroid hormone content, after the development of phase 2 thyroid follicles which might indicate pulsatile release of T4 and T3.

Effect of okadaic acid and calyculin-A, two protein phosphatase inhibitors, on thyrotropin-stimulated triiodothyronine secretion in cultured sheep thyroid cells.

We have studied the effect of two protein phosphatase inhibitors on thyrotropin (TSH)-stimulated triiodothyronine (T3) production by sheep thyroid cells grown in primary culture. Incubation of sheep thyrocytes with okadaic acid (OA) and calyculin-A (CL-A), two potent inhibitors of type 1 (PP1) and type 2A (PP2A) protein phosphatases, resulted in an increase of TSH-stimulated T3 production. This effect was detected using concentrations as low as 0.1 pM with OA and 1 fM with CL-A. An inhibitory effect on T3 production, due to cellular death, was observed with 6 nM OA and 1 nM CL-A. In the absence of TSH, OA or CL-A had no effect on T3 production by thyrocytes. Forskoline (10 microM), an activator of adenylate cyclase, increased the basal and TSH-stimulated T3 release by sheep thyroid cells; this effect was increased by OA in cells grown in the basal state but not in the presence of TSH. These results suggest that the marine toxins OA and CL-A, two potent inhibitors of PP-1 and PP-2A, have significant stimulatory effects on T3 secretion promoted by TSH and FK. These observations indicate that these proteins could be important mediators of thyroid hormone production.

Impaired thyroid function in murine toxoplasmosis.

A decline in serum thyroxine (T4) occurs in Nya: NYLAR female mice infected with Toxoplasma gondii. To ascertain whether the hypothyroxinaemia might be the result of primary thyroid dysfunction, 2 parameters of thyrofollicular cell function were monitored to determine (a) if the cell surface membrane receptors for thyroid-stimulating hormone (TSH) were operative, and (b) whether the cyclic adenosine monophosphate (cAMP)-dependent cascade of intracellular events leading to the release of T4 was responsive to exogenous cAMP. Our results indicated that both parameters were intact and functional in the infected-mouse thyrocytes. However, the elicited T4 responses were distinctly diminished in magnitude, reflecting a lack of readily available thyroidal T4 reserves. Because the continuing synthesis, storage, and release of T4 is dependent on the pulsatile stimulation of the thyroid by TSH, we suggest that the depletion of T4 reserves is likely due to perturbation of the pulsatile release of TSH from the pituitary, rather than to primary thyroid malfunction.

Salsalate administration--a potential pharmacological model of the sick euthyroid syndrome.

This study examined salsalate ingestion as a model of the sequelae of acute inhibition of thyroid hormone binding to serum protein. One dose of salsalate (60-65 mg/kg) was administered to healthy volunteers. Serum salsalate concentrations peaked at 2 h (82 micrograms/mL), then declined at 8 h to 1.2 micrograms/mL. Serum total T4 (TT4) and total T3 (TT3) concentrations declined for 4 h, then recovered by 96 h, while T4 binding protein concentrations remained unchanged. TT3 was reduced to a greater extent than TT4 between 2 h and 72 h, and serum total reverse(r)T3 (TrT3) was transiently increased at 8 h. TSH concentrations fell while TT4 and TT3 fell, then recovered while TT4, TT3, and free T3, but not free T4, were still reduced. Subsequently, TSH overshot basal levels and continued to rise after 96 h while TT4, TT3, free T4, free T3, and TrT3 were all at basal levels. We postulate that an acute release of T4 and T3 from circulating transport proteins, induced by an inhibitor of binding, can result in large and rapid redistribution of T4 and T3 into tissue compartments associated with transiently reduced peripheral tissue 5'-monodeiodination and deranged TSH regulation.

Epinephrine and norepinephrine elevate 5′-monodeiodinase activity in rainbow trout liver slices

The 5′-monodeiodinase (5′-MDA) activity was measured in liver slices that were incubated for 3 hours with epinephrine (E) or norepinephrine (NE) in order to examine the influence of these catecholamine hormones on the regulation of hepatic monodeiodination of thyroxine (T4) in rainbow trout. Both E and NE induced a dose-dependent increase in 5′-MDA activity and in addition, E stimulated the release of T3 into the medium. In liver slices taken from trout that had been treated with the β-adrenoceptor inhibitor propranolol, the response to both E and NE was attenuated. The findings provide evidence of an action of these catecholamine hormones on the peripheral regulation of T3 production, and suggest that the control operatesvia the β-adrenoceptors.

Interleukin-6 (IL-6) inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles.

Interleukin-6 (IL-6), a pleiotropic cytokine, is postulated to be involved in the pathogenesis of sick euthyroid syndrome, although the direct in vitro effects of IL-6 on human thyroid function are controversial. Because IL-6 signal can be transduced when the complex of IL-6 and soluble IL-6 receptor (sIL-6R) binds to gp 130, an IL-6 signal transducer, we studied the effects of IL-6 and sIL-6R on thyroid function, using human thyroid follicles obtained from patients with Graves' disease. IL-6 alone had no inhibitory effect on TSH-induced thyroid function (125I incorporation and organic 125I release), even at supraphysiological concentrations. However, in the presence of physiological concentrations of sIL-6R (100 ng/ml), IL-6 inhibited thyroid function dose dependently and completely, accompanied with the decreased ratio of 125I-T3/125I-T4 not only in the thyroid follicles but also in the culture medium. Thyroid follicles did not secrete sIL-6R but produced IL-6 constitutively. Consistent with these findings, sIL-6R inhibited thyroid function slightly at high concentrations. Furthermore, RT-PCR analyses revealed that human thyroid follicles expressed the messenger RNAs for IL-6 and gp130 but scarcely messenger RNA for IL-6R. These in vitro findings suggest that IL-6 alone hardly affects thyroid function in thyroid follicles in which IL-6R gene is scarcely expressed. However, because sIL-6R is present abundantly in serum, IL-6 in vivo would be capable of inhibiting the synthesis and release of T4 and, to a greater extent, T3 from the thyroid gland. These in vitro findings are at least partly related to the development of sick euthyroid syndrome.

The type-1 repeats of thyroglobulin regulate thyroglobulin degradation and T3, T4 release in thyrocytes

Thyroglobulin (Tg) proteolytic steps are central phenomena in Tg processing and thyroid hormone release in thyrocytes. Based on recent literature data, we propose that the type-1 repetitive units present in the Tg sequence could act as binders and reversible inhibitors of the proteases implicated in Tg processing. The pH-dependent interactions of proteases with the repeats could permit (i) protection from degradation of low iodinated Tg to be recycled; (ii) restriction of early proteolytic attacks to N- and C-terminal hormone formation sites; (iii) increase of the half-time of acidic proteases necessary for the final, extensive degradation steps of Tg.

Dissociation between serum interleukin-6 rise and other parameters of disease activity in subacute thyroiditis during treatment with corticosteroid.

Increased serum interleukin-6 (IL-6) concentrations have recently been reported in patients with subacute thyroiditis, possibly because of cytokine release from damaged thyroid cells. To investigate the changes in serum IL-6 concentrations in subacute thyroiditis during treatment with corticosteroid, serum IL-6 concentrations were determined by an enzyme-linked immunosorbent assay method in five patients with subacute thyroiditis. Serum IL-6 concentrations were increased moderately, and simultaneously, serum levels of T4, thyroglobulin, and C-reactive protein and erythrocyte sedimentation rate were increased markedly. The treatment with prednisolone rapidly and progressively decreased serum levels of thyroglobulin, T4, and C-reactive protein and the erythrocyte sedimentation rate. In contrast, serum IL-6 concentrations increased markedly 7 days after the treatment with prednisolone in all five patients and two of five patients showed further increases in serum IL-6 concentration on the 17th day. The rise in serum IL-6 levels in untreated patients with subacute thyroiditis in this study is compatible with previous reports. The rise in serum IL-6 levels after treatment with corticosteroid in subacute thyroiditis may reflect the dissociation between the persistent release of IL-6 from the damaged thyroid cells, immediate inhibition of secondary inflammatory reactions by corticosteroid, and the release of thyroglobulin and T4 from performed colloid stores in follicular lumen destroyed by subacute thyroiditis.

Analytical performance of free and total thyroxine assays

Excessive bias and imprecision are major analytical problems associated with some assays for free and total thyroxine (T4). Bias in free T4 methods is largely proportional to variations in serum T4 binding. In direct methods, this is attributable to requirements for substantial quantities of protein-bound T4 to replace analytical losses of free T4. In some total T4 methods, bias is inversely proportional to the amount of serum T4 binding and is attributable to the incomplete release of serum protein-bound T4. In others, bias is fixed and attributable to inaccurate calibration. Manufacturers should report the bias in their methods. Calibrations should be standardized. Imprecision varies widely among methods, but is generally less for total T4 methods than for free T4 methods. A consensus on quantitative analytical performance goals for free and total T4 methods would be helpful. Here, performance goals are proposed, based in part on the best achievements of current methods.

Thyroid glands: novel sites of growth hormone action.

Thyroid hormones inhibit the synthesis and release of GH in avian species. This may represent a feedback mechanism, since GH enhances the peripheral production of tri-iodothyronine (T3). The possibility that GH may also have direct effects on thyroidal function was therefore investigated. The basal and thyrotrophin-induced release of thyroxine (T4) from incubated chicken thyroid glands was not enhanced, however, in the presence of chicken GH. Contrarily, GH impaired T4 release in a dose-related way. These actions were probably mediated by specific receptors, since binding sites for radiolabelled GH were demonstrated on the plasma membranes of chicken thyroid glands. Expression of the GH receptor gene in these tissues was also demonstrated using a cRNA probe for the rabbit liver GH receptor, which specifically hybridized with RNA moieties of 4.4 kb, 2.7 kb and 1.0 kb. Moreover, reverse transcription of thyroidal RNA and its amplification in the presence of 3'- and 5'-oligonucleotide primers coding for the extracellular or intracellular domains of the GH receptor generated electrophoretically separable fragments of 500 bp and 800 bp respectively, as would be expected from analysis of the hepatic GH receptor cDNA sequence. Digestion of the 500 bp fragment with NcoI or EcoRI also produced moieties of expected size (350 bp and 150 bp or 325 bp and 175 bp respectively), as did BamHI or HaeIII digestion of the 800 bp fragment (yielding fragments of 550 bp and 275 bp or 469 bp and 337 bp respectively). Translation of the GH receptor mRNA was also indicated by the immunocytochemical demonstration of GH receptors in thyroid follicular and parafollicular cells, using a specific polyclonal antibody raised against the chicken GH-binding protein. These results therefore provide evidence, for the first time, of GH receptor gene expression in thyroid tissue and the translation of functional GH receptors in thyroid glands. These results also demonstrate differential effects of GH on the extracellular concentrations of T3 and T4, which may permit subtle regulation within the somatotroph-thyroid axis.

Effects of a furan fatty acid and indoxyl sulfate on thyroid hormone uptake in cultured anterior pituitary cells

A furan fatty acid, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) and indoxyl sulfate (Indox) accumulate in serum of uremic patients and inhibit the active uptake of thyroxine (T4) into hepatocytes. We tested the effects of CMPF and Indox on the uptake of [125I]triiodothyronine (T3) and [125I]T4 and thyroid-stimulating hormone (TSH) release in anterior pituitary cells. Pituitary cells (500,000/well) were cultured for 3 days in medium with 10% fetal calf serum. Experiments were performed at 37 degrees C in the same medium with 0.5% bovine serum albumin (BSA; [125I]T3 uptake and TSH secretion) or 0.1% BSA ([125I]T4 uptake). The 15-min uptake of [125I]T3 amounted to 0.074 +/- 0.003 fmol/pM free T3 (n = 23) and that of [125I]T4 to 0.033 +/- 0.002 fmol/pM free T4 (n = 32). Preincubation (30 min) and incubation (15 min) with CMPF (20-200 microM) did not alter the uptake of [125I]T3 but reduced [125I]T4 uptake by 27% (P < 0.05) at the highest concentration tested. Indox (40-400 microM) did not affect the uptake of [125I]T3 or [125I]T4. CMPF (40 microM) and Indox (80 microM) did not directly affect the basal or thyrotropin-releasing hormone (TRH)-induced TSH release nor interfere with the effect of 10 nM T3 on TRH-induced TSH release. In conclusion, the absence of inhibitory effects of CMPF or Indox on thyroid hormone uptake by pituitary cells suggests that the transport mechanism is regulated differently compared with that in hepatocytes and underscores the significance of the thyroid hormone carriers for the intracellular availability of T3.

Pituitary and peripheral thyroid hormone responses to thyrotropin-releasing hormone during sustained sleep deprivation in freely moving rats.

Sleep deprivation is associated with poor cognitive ability and impaired physical health, but the ways in which the brain and body become compromised are not understood. In sleep-deprived rats, plasma total T4 and T3 concentrations decline progressively to 78% and 47% below baseline values, respectively, brown adipose tissue 5'-deiodinase type II activity increases 100-fold, and serum TSH values are unknown. The progressive decline in plasma thyroid hormones is associated with a deep negative energy balance despite normal or increased food intake and malnutrition-like symptoms that eventuate in hypothermia and lethal systemic infections. The purpose of the present experiment was to evaluate the probable causes of the low plasma total T4 during sleep deprivation by measuring the free hormone concentration to minimize binding irregularities and by challenging the pituitary-thyroid axis with iv TRH to determine both 1) the pituitary release of TSH and 2) the thyroidal response of free T4 (FT4) and free T3 (FT3) release to the TSH increment. Sleep-deprived rats were awake 91% of the total time compared with 63% of the total time in yoked control rats and 50% of the total time during the baseline period. Cage control comparison rats were permitted to sleep normally. Sustained sleep deprivation resulted in a decline from baseline in plasma FT4 of 73 +/- 6% and FT3 of 45 +/- 12%, which were similar to the declines in total hormone concentrations observed previously; nonstimulated TSH was unchanged. In the yoked and cage control groups, FT4 also declined, but much less than that of the sleep-deprived group. The relative changes in free compared with total hormone concentrations over the study were also less parallel than those in the sleep-deprived group. The plasma TSH response to TRH was similar in all groups across experimental days. The plasma FT4 and FT3 concentrations in sleep-deprived rats increased after TRH-stimulated TSH release to an extent comparable to control values. Taken together, low basal FT4 and FT3 hormone concentrations and unchanged TSH and thyroidal responses to TRH suggest a pituitary or hypothalamic contribution to the hypothyroxinemia during sleep deprivation.

Extrathyroidal release of thyroid hormones from thyroglobulin by J774 mouse macrophages.

Thyroglobulin appears in the circulation of vertebrates at species-specific concentrations. We have observed that the clearance of thyroglobulin from the circulation occurs in the liver by macrophages. Here we show that the thyroid hormones T3 and T4 were released by incubation of mouse macrophages (J774) with thyroglobulin. Thyroid hormone release was a fast process, with an initial rate of approximately 20 pmol T4/mg per min and approximately 0.6 pmol T3/mg per min, indicating that macrophages preferentially release T4. The bulk of released thyroid hormones appeared after 5 min of incubation of macrophages with thyroglobulin, whereas degradation of the protein was detectable only after several hours. During internalization of thyroglobulin, endocytic vesicles and endosomes were reached at 5 min and lysosomes at 60 min. T4 release started extracellularly by secreted proteases and continued along the endocytic pathway of thyroglobulin, whereas T3 release occurred mainly intracellularly when thyroglobulin had reached the lysosomes. This shows that the release of both hormones occurred at distinct cellular sites. Our in vitro observations suggest that macrophages in situ represent an extrathyroidal source for thyroid hormones from circulating thyroglobulin.