Colloidal aggregates of insoluble inclusions in human goiters.

To shed some light on the physicochemical properties of the thyroid follicular colloid, we have screened retrospectively the autoradiographs of 60 human nodular goiters labeled 17 h preoperatively with 100 microCi 125I for evidence of colloid compartmentalization. In 87% (52/60) of all goiters examined, sporadic or multiple colloidal inclusions ('colloid stones') not mixing with newly labeled Tg were detected. The detailed analysis of 17 goiters revealed a mean incidence of 0.09+/-0.11 'colloid stones' of variable size per follicle ranging from 0.02+/-0.01 (10) to 0.43+/-0.09 (5) (mean values +/- S.D., number of sections examined in brackets). In this study we did not find a clear-cut association of incidence of 'colloid stones' with sex, age or nosologic group (hyperthyroid, preclinically hyperthyroid, euthyroid). The existence of different colloidal compartments as demonstrated in this and other studies is of considerable importance for thyroid function, interpretation of iodine kinetics, and studies on the role of iodine on growth and function of the thyrocytes. Different thyroidal iodine compartments could well be of functional relevance, for example in the adaptation of thyroid hormone secretion to antithyroid drugs or in severe and prolonged iodine deficiency, when very slow compartments become an important source of minimal quantities of iodine and thyroid hormone. 'Colloid stones', for example, may well explain the repeatedly observed, surprisingly large total iodine store in human endemic goiters, even in the presence of severe iodine deficiency. It is evident that the existence of multiple iodine compartments and, in particular, of particulate slow-turnover pools complicates the interpretation of total glandular iodine measurements with modern techniques such as X-ray fluorescence and positron emission tomography.     

Differences in iodinated peptides and thyroid hormone formation after chemical and thyroid peroxidase-catalyzed iodination of human thyroglobulin

The distribution of iodine among the polypeptides of human goiter thyroglobulin (Tg) was examined. Tg was iodinated in vitro with ¹³¹I to levels of 2 to 84 gram atoms (g.a.)/mol using thyroid peroxidase (TPO) or a chemical iodination system. The samples were reduced, alkylated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two low-molecular-weight peptides appeared preferentially in radioautograms of the sodium dodecyl sulfate (SDS) gels of TPO-iodinated samples. Iodination of these peptides increased sharply in the TPO-treated Tg as the level of total iodine/ molecule rose. Radioiodine was incorporated into these same gel regions in the chemically treated Tg, but only after much higher levels of total iodination were reached. Differences in iodoamino acid distribution were also noted between the chemically and enzymatically iodinated thyroglobulins. In the chemically iodinated samples, little thyroxine (T₄) was synthesized, even at high iodine levels. In the TPO-treated samples only small amounts of T₄ were seen below 14 g.a. total I/mol, while at or above that level of iodination T₄ formation increased sharply. To examine the coupling process, Tg was chemically iodinated, excess I^? removed, and the samples treated with TPO and a H₂O₂-generating system in the absence of iodide. Radioautograms obtained from SDS-polyacrylamide gels of reduced and alkylated protein from such coupling assays showed an increase in the level of iodine in the low-molecular-weight peptides after TPO treatment. Thyroxine production also increased with TPO treatment. The addition of free DIT (a known coupling enhancer) to the [¹³¹I]Tg/TPO incubation increased both the production of T₄ and the amount of iodine in the smaller polypeptides. Two-dimensional maps prepared from CNBr-digested TG showed differences between the coupled and uncoupled samples. Our observations confirm the importance of the lowmolecular-weight peptides derived from Tg in thyroid hormone synthesis. At total iodine levels above 14 g.a./mol Tg in enzymatically treated samples there is selective incorporation of iodine into both the low-molecular-weight polypeptides and into thyroid hormone.     

Epidermal growth factor (EGF) inhibits stimulated thyroid hormone secretion in the mouse

It is known that epidermal growth factor (EGF) inhibits iodide uptake in the thyroid follicular cells and lowers plasma levels of thyroid hormones upon infusion into sheep and ewes. In this study, the effects of EGF on basal and stimulated thyroid hormone secretion were investigated in the mouse. Mice were pretreated with 125I and thyroxine; the subsequent release of 125I is an estimation of thyroid hormone secretion. It was found that basal radioiodine secretion was not altered by intravenous injection of EGF (5 micrograms/animal). However, the radioiodine secretion stimulated by both TSH (120 microU/animal) and vasoactive intestinal peptide (VIP; 5 micrograms/animal) were inhibited by EGF (5 micrograms/animal). At a lower dose level (0.5 microgram/animal), EGF had no influence on stimulated radioiodine secretion. In conclusion, EGF inhibits stimulated thyroid hormone secretion in the mouse.     

Effect of potassium iodide and perchlorate on the process of thyroid hormone secretion]

The influence of potassium iodide and perchlorate on the parameters characterizing the thypoid hormones secretion, such as the cAMP level in the gland tissue and the number of intracellular colloid droplets under condition of stimulation by thyrotropic hormone was studied. It was shown that the abovementioned parameters were depressed by an excess of iodide, but perchlorate administration prevented the inhibitory effect of iodide. The results obtained favour the conception on the sensitivity of the thyroid adenylate cyclase system to the organic iodine concentration. Apparently and excess of iodide depressed the capacity of perchlorate to influence its concentration in the gland, and thereby the process of iodine organification and of the thyroid hormone secretion maintained at the optimal leve.     

Iodinated phospholipids and the iodination of proteins of dog thyroid gland in vitro

Slices of dog thyroid gland were incubated with liposomes consisting of (125)I-labelled phosphatidylcholine (the iodine was covalently linked to unsaturated fatty acyl chains). The (125)I label of (125)I-labelled liposomes was incorporated into thyroid protein and/or thyroglobulin at a higher rate than was the (131)I label of either Na(131)I or (131)I(2). The iodine was shown to be protein-bound by the co-migration of the labelled iodine with protein under conditions where free iodine, iodide and lipid-bound iodine were removed from protein. The uptake of iodine from the iodinated phospholipid was probably due to phospholipid exchange between the iodinated liposomes and the thyroid cell membrane, since (a) (14)C-labelled phospholipid was metabolized to (14)CO(2) and (b) many lipids in the tissue slice became (14)C-labelled. A very strong inhibition of iodide ;uptake' from Na(131)I, caused by thiosulphate, produced only a minor inhibition of the incorporation of (125)I from (125)I-labelled liposomes into thyroid protein and/or thyroglobulin. This implies that free iodide may not necessarily be formed from the iodinated phospholipids before their entrance or utilization in the cell. Synthetic polytyrosine polypeptide suspensions showed some iodination by (131)I-labelled liposomes. In tissues with low tyrosine contents, such as liver and kidney, only a trace uptake was observed. Salivary gland showed some uptake. Endoplasmic reticulum of thyroid gland showed a higher iodine uptake than that of the corresponding plasma membranes. These experiments, together with the demonstration of the diet-dependent presence of iodinated phospholipids in dog thyroid, leads us to suggest that iodination of the membrane phospholipids of thyroid cells may be directly or indirectly involved at some stage in the synthesis of thyroglobulin, or exists as a scavenger mechanism, to re-utilize and/or recover released iodine from unstable compounds inside the thyroid cell.     

Free diiodotyrosine effects on protein iodination and thyroid hormone synthesis catalyzed by thyroid peroxidase.

Free diiosotyrosine exerts two opposite effects on the reactions catalyzed by thyroid peroxidase, thyroglobulin iodination and thyroid hormone formation. 1. Inhibition of thyroglobulin iodination catalyzed by thyroid peroxidase was observed when free diiodotyrosine concentration was higher than 5 muM. This inhibition was competitive, suggesting that free diiodotyrosine interacts with the substrate site(s) of thyroid peroxidase. Free diiodotyrosine also competively inhibited iodide peroxidation to I2. 2. Free diiodotyrosine, when incubated with thyroid peroxidase in the absence of iodide was recovered unmodified; in the presence of iodide an exchange reaction was observed between the iodine atoms present in the diiodotyrosine molecule and iodide present in the medium. Using 14C-labelled diiodotyrosine, 14C-labelled non-iodinated products were also observed, showing that deiodination occurred as a minor degradation pathway. However, no monoiodo[14C]tyrosine or E114C]tyrosine were observed. Exchange reaction between free diiototyrosine and iodide is therefore direct and does not imply deiodination-iodination intermediary steps. Thyroglobulin inhibits diiodotyrosine-iodide exchange and vice versa, again suggesting competition for both reactions. These results support, by a different experimental approach, the two-site model for peroxidase previously described by us in this journal. 3. Free diiodotyrosine when present at a very low concentration, 0.05 muM, exerts a stimulatory effect on throid hormones synthesis. The relationship between diiodotyrosine concentration and thyroid hormone synthesis give an S-shaped curve, suggesting that free diiodotyrosine acts as a regulatory ligand for thyroid peroxidase. Evidence is also presented that free diiodotyrosine is not incorporated into thyroid hormones. Therefore, thyroid peroxidase catalyzes only intra-molecular coupling between iodotyrosine hormonogenic residues. 4. Finally, although no direct proof exists that these free diiodotyrosine effects upon thyroglobulin iodination and thyroid hormone synthesis are physiologically significant, such a possibility deserves further investigation.     

The follicular iodide pool as a two-compartment system: evidence from the unstimulated thyroid gland

The isotopic equilibrium method was used for a 60-day period to follow iodine turnover in control and hypophysectomized rats. After the suppression of TSH by hypophysectomy, only the captured iodide is used for iodination of thyroglobulin, the iodide recycling being abolished. In unstimulated gland, two metabolically distinct iodide compartments do exist which differ either in their chemical form or in their morphological distribution, or both.     

Thyroid hormone residues are released from thyroglobulin with only limited alteration of the thyroglobulin structure

We have tried to characterize thyroglobulin (Tg) degradation products in purified pig thyroid lysosomes to determine whether the release of thyroid hormone residues from Tg involves a random proteolytic attack or discrete and selective cleavage reactions. The intralysosomal soluble protein fraction was prepared by osmotic pressure-dependent lysis of lysosomes purified by isopycnic centrifugation on Percoll gradients. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a fraction of Tg (5-10% of total lysosomal protein) with the same molecular weight as that of the intact Tg subunit. This high molecular weight Tg was the only intralysosomal species detected by Western blot using antipig Tg antibodies. In nondenaturing conditions, lysosomal Tg (LTg) identified by radioimmunoassay was in the form of a dimer with a sedimentation coefficient lower than that of either iodinated Tg (colloid Tg) or noniodinated Tg (microsomal Tg). LTg had a lower iodine content than colloid Tg:9-12 versus 39-42 iodine atoms/molecule. Pronase hydrolysates of LTg did not contain any 3,5,3',5'-tetraiodo-L-thyronine or 3,3',5-triiodo-L-thyronine residues detectable by reverse-phase high pressure liquid chromatography; iodine present in LTg was in the form of iodotyrosines. Under reducing conditions, LTg almost completely disappeared and gave rise to various polypeptides of smaller size. These results suggest that Tg transferred to lysosomes is subjected to selective proteolytic cleavage reaction(s) that release thyroid hormone residues. This early step would lead to the formation of hormone-depleted Tg molecules that are cleaved at discrete sites, the resulting polypeptides remaining bound through disulfide bonds to yield Tg molecules with an apparently normal size and a slightly altered structure.     

The importance of thyroglobulin structure for thyroid hormone biosynthesis.

Thyroglobulin (Tg) is the most important protein in the thyroid because it provides the matrix for thyroid hormone biosynthesis. Here we review experimental work, principally from our laboratory, on the relationship between Tg structure and hormonogenesis. Early work showed that Tg's most important hormonogenic site was located in a fragment of approximately 26 kDa obtained on chemical reduction. With the establishment of the cDNA sequence of Tg, this and other major sites could be localized within Tg's polypeptide chain. The four major hormonogenic sites, designated A, B, C, and D, are located respectively at tyrosyls 5, 2553, 2746, and 1290. In most species, site A accounts for about 40% of Tg's hormone, and site B for about 25%. Site C is associated with increased T3, at least in some species. Site D is prominent in guinea pigs and rabbits, and TSH favors hormonogenesis at it in these species. Sequential iodination of low iodine human Tg shows three consensus sequences associated with early iodination and with T4 formation. Recent work has identified Tyr130 in beef Tg as donor of an outer iodothyronine ring, most likely to Tyr5, the most important hormonogenic site. In addition to its biochemical importance, Tg has clinical interest in familial goiter and autoimmune thyroid disease. Further elucidation of Tg structure and its relation to thyroid hormone synthesis will contribute to thyroid physiology and to its clinical application.     

Critical role of iodination for T cell recognition of thyroglobulin in experimental murine thyroid autoimmunity

We have used two clonotypically distinct thyroglobulin (Tg)-specific, I-Ak restricted monoclonal T cell populations to investigate the role of thyroid peroxidase-catalyzed iodination in Tg recognition by autoreactive T cells. The results showed that these T cells could recognize Tg only it it was sufficiently iodinated. Unlike normal mouse Tg, noniodinated mouse Tg was unable to induce significant thyroid lesions but could trigger the production of Tg autoantibodies. In these experiments, the importance of T cell recognition of iodination-related epitopes was emphasized by the inability of serum antibodies to distinguish Tg on the basis of iodine content, whether they were induced with normal or noniodinated Tg. Therefore, thyroid peroxidase-dependent modification of Tg would appear to be central to its recognition by autoreactive T cells and hence its capacity to induce autoimmune thyroid lesions.     

The subcellular distribution of 32P-labelled phospholipids, 32P-labelled ribonucleic acid and 125I-labelled iodoprotein in pig thyroid slices. Effect in vitro of thyrotrophic hormone and dibutyryl-3′,5′-(cyclic)-adenosine monophosphate

1. The incorporation in vitro of [(32)P]phosphate into phospholipids and RNA and of [(125)I]iodide into protein-bound iodine by pig thyroid slices incubated for up to 6hr. was studied. The subcellular distribution of the labelled products formed after incubation with radioactive precursor in the nuclear, mitochondrial, smooth-microsomal, rough-microsomal and cell-sap fractions was also studied. 2. Pig thyroid slices actively took up [(32)P]phosphate from the medium during 6hr. of incubation; the rate of incorporation of (32)P into phospholipids was two to five times that into RNA. 3. The uptake of [(125)I]iodide by the slices from the medium was rapid for 4hr. of incubation, 6-10% of the label being incorporated into iodoprotein. 4. Much of the (32)P-labelled phospholipid accumulated in mitochondria and microsomes, whereas the nuclear fraction contained most of the (32)P-labelled RNA. After 2hr. of incubation most of the (32)P-labelled cytoplasmic RNA accumulated in the rough-microsomal fraction. The major site of localization of proteinbound (125)I was the smooth-microsomal fraction, and gradually increasing amounts appeared in the soluble cytoplasm fraction, suggesting a vectorial discharge of [(125)I]iodoprotein (presumably thyroglobulin) from smooth vesicles into the colloid. 5. The addition of 0.1-0.4 unit of thyrotrophic hormone/ml. of incubation medium markedly enhanced the accumulation of (32)P-labelled phospholipids in the microsomal fractions and to a much smaller extent that of (32)P-labelled RNA without any increase in the total uptake of the label. Almost simultaneously the hormone increased the uptake of [(125)I]iodide by the slices and enhanced the accumulation of protein-bound (125)I in the smooth-microsomal fraction. 6. As a function of time of incubation, thyrotrophic hormone had a biphasic effect on [(125)I]iodide uptake and protein-bound (125)I formation, the stimulatory effect being reversed after 4hr. of incubation. 7. 6-N-2'-O-Dibutyryl-3',5'-(cyclic)-AMP, but not 3',5'-(cyclic)-AMP or 5'-AMP, mimicked the action of thyrotrophic hormone on iodine uptake as well as on iodination of protein. On the other hand, the mimicry by 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP of the stimulatory effect of thyrotrophic hormone on the formation of labelled thyroid phospholipids and RNA was only an apparent one resulting from an enhanced uptake of [(32)P]phosphate. 8. It is concluded that thyrotrophic hormone causes a co-ordinated increase in the formation or accumulation of phospholipids, RNA and iodoprotein associated with the endoplasmic reticulum, and that 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP mimics the more rapid effects of thyrotrophic hormone on transport and metabolic functions of thyroid cells, but does not influence their slower biosynthetic responses to the hormone.     

Iodoamino acid composition of rat thyroglobulin of varying total iodine concentration and fractions isolated by isopycnic ultracentrifugation]

Iodoaminoacid content (iodothyronines, T3 and T4, and iodotyrosines, MIT and DIT) has been determined in enzymatic hydrolysates of thyroglobulin Tg 19S of different iodine content (0.3-0.9%) isolated from equilibrium labeled rats. Preparative equilibrium centrifugation in RbCl density gradients of pure thyroglobulin was used to obtain protein fractions of largely different iodine content (0.2-1.2% I). Thin layer chromatography of total hydrolysates demonstrated that the distribution of iodoaminoacids depends on the total iodine content of each fraction. It is concluded, in agreement with previous results, that the native structure of Tg is an important factor in the regulation of hormone biosynthesis and that even at low iodination levels of Tg. T3 and T4 are synthesized.     

Changes in iodine mapping in rat thyroid during the course of iodine deficiency: imaging and relative quantitation by analytical ion microscope

The analytical ion microscope (AIM) makes possible imaging and relative quantitation of multiple stable or labeled elements on an even tissue section, according to their mass. The purpose of this work was to follow at the rat thyroid follicle level the changes in 127I mapping during low iodine diet (LID) in relation to the ability of thyroid to pick up radioiodine (129I) and to synthesize Tg from its precursor, 2H-labeled leucine. The overall picture of images and countings of 127I shows a progressive decrease of the luminal iodine concentration which on day 80 was 10-fold lower than that of control value. In control rat thyroid cell, concentration was 10-fold lower than that of follicular lumina and was unchanged until 35 days, but the size of the cytoplasmic compartment increased, suggesting a redistribution of iodine stores between thyroid cells and follicular lumina. 129I was always found in colloid as well as in cells at all stages. After 35 days of LID, cytoplasmic and luminal radioiodine concentrations decreased. In control rats, [2H]leucine was found mainly in the cells. During LID its localization was evidenced progressively in most of the lumina. The most striking fact was the presence up to 35 days of some large residual follicles with high 127I concentration and low 129I and 2H incorporation. These data demonstrate the follicular heterogeneity of thyroid response to progressive chronic TSH stimulation induced by LID.     

Manganese ion as a goitrogen in the female mouse

Effect of excessive ingestion of manganese (Mn) on the mouse thyroid was assessed under the conditions of normal intake of iodide. Female mouse thyroids were enlarged after 7 weeks of administration of 200 mg/l MnCl2 X 4H2O in drinking water; 2.74 +/- 0.25 mg for control (N = 56), and 3.31 +/- 0.28 mg for Mn-treated group (N = 85) (p less than 0.001). In contrast, male mouse thyroids never became goitrous following this treatment. Manganese was goitrogenic to the castrated male mouse, but it had no effect on the testosterone-treated castrated male mouse, indicating the involvement of androgen in goiter formation. Oral administration of Mn did not severely affect blocked T/S of 125I or iodine metabolism in the thyroid. A morphological study, however, revealed that the epithelial cell in the Mn-treated mouse thyroid became flatter than that of the control. The lumens were filed with colloid in Mn-treated female mouse thyroid. The serum levels of thyroxine (T4), but not triiodothyronine (T3), were slightly reduced by Mn. These informations suggest that Mn can be a mild goitrogen for the female mouse and that the etiology of goiter formation can be interpreted by retention of colloid in the lumen.     

Effects of microtubule inhibitors and cytochalasin B on thyroid metabolism in vitro.

Mitotic spindle inhibitors (colchicine, vinblastine, vincristine, 020, ethanol) and cytochalasin B inhibit the phagocytosis of colloid by thyroid cells and the secretion of thyroid hormones. This inhibition has been linked to interferences with the microtubular microfilament system of the follicular cell. In order to test the possibility of using such inhibitors to selectively block secretion, the action of suppressing or highly inhibitory concentrations on other metabolic parameters has been studied on dog thyroid slices in vitro: glucose oxidation, lactate formation, iodide binding to protein, cyclic 3'5' AMP accumulation. It is shown that at a concentration of 10 mM colchicine is entirely non specific as it greatly inhibits all facets of metabolism and all the stimulatory effects of cyclic 3'5' AMP and thyrotropin. The other mictrotubule inhibitors, although affecting thyroid metabolism in various ways were more specified. The enhancement by vineblastine of glucose oxidation ald iodine binding to proteins suggests an activation of they thyroid H2O2 generating system. D2O on the other hand selectively inhibits secretion and the binding of iodide to proteins. Cytochalasin B, presumably by inhibiting hexose transport, decreased glycolysis and the uptake of iodide. However this effect cannot account for the complete inhibition of thyroid secretion.     

Similar serum concentrations of thyroid hormones in two geographically separate populations on disparate iodine intake.

Serum thyroid hormones were measured in Montreal, Canada (urinary iodine 446 +/- 164 micrograms/day) and Zagreb, Yugoslavia (urinary iodine 108 +/- 32 microgram/day). The serum concentrations of thyroxine and triiodothyronine in the two populations were almost identical. We conclude that dietary iodine, within accepted normal limits, is not a factor in determining serum thyroid hormone levels. The wide differences in reported serum triiodothyronine concentrations are related to methodological problems.     

Role of immune complexes in thyroid diseases]

To elucidate the immune complex (thyroglobulin-antibody, Tg-Ab) role in thyroid hormone formation, in vitro iodination of Tg--AB immune complex, bovine intact Tg and that of the patient with euthyroid goiter and partially purified AB to Tg were studied. We compared the amount of iodamine acids (MIT, DIT, T4/mole of Tg) forming during iodination in immune complex with other samples. The results suggest that Tg being in complex with antibodies (up to 30-40 mole of antibodies/mole of Tg) is iodinated forming the enough amount of MIT, decreased amount of DIT and T4. A MIT fraction increase is connected with additional iodination of complex antibodies. We suggest that such processes may take place in the patient body and be involved in pathogenesis of thyroid disease.     

Hormonogenesis in thyroid cells cultured on porous bottom chambers

Different processes implied in thyroid hormonogenesis (thyroglobulin, thyroperoxidase and hydrogen peroxide generating system expressions) and their regulation by TSH and iodide have been studied using porcine thyroid cells cultured in porous bottomed chambers. This system allowed to reproduce the functional bipolarity. Cells form a tight and polarized monolayer. Both apical and basolateral poles of epithelial cells were independently accessible and the cell layer separated two compartments which can contain different media. A major polarized secretion of thyroglobulin into the apical compartment was observed; it was increased in the presence of TSH as well as the thyroglobulin synthesis and mRNA level. These TSH effects were the consequence of adenylcyclase stimulation. Active transport of iodide, iodination of thyroglobulin and hormonosynthesis took place only in the presence of TSH. These steps occurred at the apical pole of cells. In the culture chamber system, thyroglobulin was weakly iodinated (6 atoms of iodide per mole of thyroglobulin; in vivo up to 40 atoms per mole) but hormonogenesis efficiency was close to this one observed in vivo (40%). Iodide concentrations higher than 0.5 µM daily added to the basal medium inhibited iodination of thyroglobulin and hormonosynthesis. Some components contained in culture media were inhibitors for iodination when they were present in the apical medium such as vitamins, amino acids and phenol red. The culture system appears to be interesting for pharmacological and toxicological studies.