Physical health impairment and depression among older adults

We studied the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and interferon-gamma (IFN-gamma) on the function of thyroid cells and pituitary thyrotrophs. In FRTL-5 rat thyroid cells, both human and murine TNF-alpha inhibited basal and TSH-stimulated [125I]iodide transport. IL-1 shared this action with TNF-alpha, but was less potent. IL-1 and IFN-gamma did not cause a further reduction of TNF-alpha-induced inhibition of [125I]iodide transport. TNF-alpha, phorbol ester 12-myristate 13-acetate (PMA), and calcium ionophore (CI) A23817 all inhibited [125I]iodide transport, but high doses of PMA and CI also blocked the inhibitory action of TNF-alpha on [125I]iodide transport. Inhibition of protein kinase A and protein kinase C by H7 or HA inhibited TSH-stimulated iodide transport, but did not block the TNF-alpha action, suggesting that the mechanism of TNF-alpha action on thyroid cells is independent of protein kinase A and C. In pituitary cells, both human and murine TNF-alpha did not affect basal TSH secretion, but TNF-alpha reduced TRH-stimulated TSH secretion. This study provides further in vitro evidence that TNF-alpha inhibits the function of the hypothalamus-pituitary-thyroid axis acting directly on both the pituitary and thyroid glands.     

Inhibition of thyroglobulin biosynthesis and degradation by excess iodide. Synergism with lithium

Lithium and excess iodide inhibit the release of thyroid hormone from preformed stores. We thus tested the hypothesis that this was due to an inhibition of thyroglobulin breakdown. Rats were pre-treated with propyl-thiouracil (PTU) for 3 weeks in order to deplete their thyroids of thyroglobulin. While the PTU was continued, lithium chloride (0.25 mEq./100 g weight) or potassium iodide (3 mg per rat) were injected every 12 h for d days. Thereafter the thyroglobulin content in thyroid gland homogenates was measured. PTU pre-treatment lowered the thyroglobulin content from 4.21 to 0.22 mg/100 mg gland. Lithium caused a marked re-accumulation of thyroglobulin to 0.60 mg/100 mg within 3 days. While iodide alone had only a borderline effect, it markedly potentiated the action of lithium and a combination of the two drugs increased the thyroglobulin content to 1.04 mg/100 mg. Thyroxine was injected into similarly pre-treated animals to suppress secretion of thyrotrophic hormone. This markedly inhibited the proteolysis of thyroglobulin and 1.3 mg/100 mg gland accumulated after 3 days. Excess iodide, given in addition to thyroxine, decreased the amount of thyroglobulin accumulated to 0.75 mg/100 mg gland. To study whether this could be explained by an inhibitory action of iodide on thyroglobulin biosynthesis, thyroid glands from animals treated with excess iodide were incubated in vitro in the presence of 0.2 mM iodide for 3 h. Iodide decreased the incorporation of radioactive leucine into total thyroidal protein and into thyroglobulin by 25 and 35% respectively. Iodide did not inhibit protein synthesis in the kidney, liver or muscle tissue. Thus, large doses of iodide selectively inhibit thyroglobulin biosynthesis.     

Changes in the polypeptide assembly of guinea pig thyroglobulin induced by thyrotropin-regulated thyroid activity

We have previously reported that the relative proportion of three polypeptide chains in guinea pig thyroglobulin is closely related to the iodine content of the protein. The present work demonstrates that it is not the iodine content per se but, rather, TSH-regulated thyroid activity which modulates the substructure of thyroglobulin. In a first set of experiments, the impact of TSH stimulation on sodium dodecyl sulfate (SDS)-induced dissociation of 19S thyroglobulin into 12S subunits was compared to that of iodination. While in control animals the ratio of 12S to 19S thyroglobulin was 48:52, it changed to 35:65 in glands strongly stimulated with TSH and blocked with MMI. This rise in the relative proportion of 19S thyroglobulin occurred despite a simultaneous drop of iodine content from 0.6% to 0.24%. It was only after TSH suppression that the well known inverse correlation between the level of iodination and dissociability reappeared. In a second set of experiments, SDS-treated thyroglobulin was fully reduced by splitting disulfide bonds with mercaptoethanol. In addition to the previously described three polypeptide chains, A, B, and C, a hitherto neglected nonreducible fraction comigrated with 19S thyroglobulin on polyacrylamide gels. Native thyroglobulin with widely varying iodine contents was obtained from unstimulated glands and from glands strongly stimulated with TSH. Drastic changes in the polypeptide chain assembly, depending on the degree of TSH stimulation but entirely independent of iodination, were observed. There was a strong negative correlation between the nonreducible 19S thyroglobulin fraction and both the B and C polypeptide chains with all experimental manipulations. We conclude that thyroglobulin substructure is highly dependent on the degree of TSH stimulation of the thyroid. TSH, through stimulation of unknown metabolic pathways, is a more important determinant of thyroglobulin substructure than the degree of iodination of the protein.     

Differential effects of protein kinase A on Ras effector pathways

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, RasS35 and RasG37, which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing RasS35, a mutant which binds Raf, or RasG37, a mutant which binds RalGDS, exhibited TSH-independent proliferation. RasS35 stimulated morphological transformation and anchorage-independent growth. RasG37 stimulated proliferation but not transformation as measured by these indices. TSH exerted markedly different effects on the Ras mutants and transiently repressed MAPK phosphorylation in RasS35-expressing cells. In contrast, TSH stimulated MAPK phosphorylation and growth in cells expressing RasG37. The Ras mutants, in turn, exerted differential effects on TSH signaling. RasS35 abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while RasG37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.     

Studies on the mechanism of action of potassium iodide on thyroid protein biosynthesis

Potassium iodide (KI) has been shown to have an antigoitrogenic action and to inhibit in vivo thyroid protein biosynthesis. Beef thyroid slices were used to clarify further the mechanism of action of KI. Incubations were performed in Krebs-Ringer-bicarbonate (KRB) buffer under 95%O2 and 5% CO2. KI caused a slight decrease in the uptake of [3H]eucine by the tissue. When labelled leucine incorporation into protein was measured it was found that 10(-6) M KI caused a marked inhibition. Increasing concentrations of KI up to 10(-3) M did not further increase this inhibition. This effect of KI was reduced by simultaneous addition of 0.5 mM KClO4 or 1 mM methylmercaptoimidazole (MMI). In several experiments it was found that equimolar amounts of thyroxine (T4) or triiodothyronine (T3) were more potent than KI in inhibiting thyroid protein biosynthesis. In double plabelled studies KI decreased [3H]leucine incorporation into thyroid soluble proteins and into immunoprecipitable thyroglobulin (T4) while it did not modify that of [14C]galactosamine. When tissue specificity was examined, KI failed to alter [3H]leucine incorporation into proteins either in the liver or in the submaxillary gland. The present results indicate that intracellular KI is necessary to exert its effect on protein synthesis, and that this effect is mediated through a organic form of iodine, probably iodothyronines. This action of KI is specific for the thyroid gland.     

Thyroglobulin assay in the follow-up of patients with differentiated thyroid carcinomas: comparison of its value in patients with or without normal residual tissue

The usefulness of serum thyroglobulin (Tg) assay in the follow-up of differentiated thyroid carcinomas has been evaluated in 109 subjects divided into two groups. Group 1 included 64 patients who had undergone total thyroid ablation. In 40 of the 41 patients in complete remission serum Tg was undetectable during replacement therapy (TSH below 5 microunits/ml). In 18 out of the 40 patients serum TG was detectable following endogenous TSH stimulation. As 83% of these patients had ectopic uptake prior to the last radioiodine treatment, this release of Tg under TSH stimulation suggests the persistence of occult neoplastic tissue. Of the other 23 patients, 20 had bone or lung metastases and 3 patients had lymph node recurrences: in all these patients, serum Tg was detectable during replacement therapy and increased after TSH stimulation. Group 2 included 45 patients in whom normal residual thyroid tissue was present at the time of the investigation. Of these, 35 patients were in apparent remission and 19 of them had detectable Tg level within the normal range. The other 10 patients had detectable metastases and in 4 of these the Tg level was also within the normal range. Thus, no conclusion can be drawn from a normal Tg level in the presence of residual thyroid tissue. Bovine TSH stimulation did not improve significantly the diagnostic value of Tg assay in this group of patients.     

Differential expression of thrombospondin, collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[125I] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450-480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [131I]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450-480 resolved to M(r) 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin I. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from M(rs) 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factors 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium.     

Long-term effect of norepinephrine on iodide uptake in FRTL-5 cells

The sympathetic nervous system plays a role in the regulation of thyroid function. In FRTL-5 rat thyroid cells, norepinephrine (NE) acutely depresses intracellular I- by increasing I- efflux. The present study was undertaken to determine the effect of NE on iodide transport after a longer time period. NE inhibited the ability of thyrotropin (TSH) to induce iodide uptake by FRTL-5 cells after 48 or 72 hours, but not after 24 hours. The effect of NE was more evident with increasing concentrations of TSH. NE did not modify the rate of I- efflux. Inhibition was associated with a decrease in the Vmax and no change in the Km for iodide influx. To determine if this was a generalized effect of NE on thyroid cell membrane, the uptake of alpha-aminoisobutyric acid (a nonmetabolizable aminoacid) and of 2-deoxyglucose was measured. NE did not inhibit TSH stimulation of the uptake of the two compounds. NE inhibited the action of dibutyryl cAMP (dbcAMP) on iodide uptake in a similar manner to TSH, but did not alter the cyclic adenosine monophosphate (cAMP) levels increased by TSH. The effects of different adrenoreceptor agonists and antagonists demonstrated that norepinephrine acts through an alpha1-adrenergic receptor.     

Effect of postoperative 131I treatment on thyroglobulin measurements in the follow-up of patients with thyroid cancer

Correlation of serum thyroglobulin (Tg) levels with recurrent cancer was performed in 200 patients who had undergone a subtotal thyroidectomy for well-differentiated thyroid carcinoma. Patients were divided into three groups: (1) those not treated postoperatively with radioactive iodine, (2) those treated with low dose (30 mCi) radioactive iodine, and (3) those treated with high dose (50-250 mCi) radioactive iodine. Tg levels proved to be reliable in detecting recurrent thyroid cancer regardless of the dose of radioactive iodine given postoperatively. These results reinforce the recommendation of using the Tg assay as the primary method of following these patients postoperatively, even when there was less than a total thyroidectomy and ablation with radioactive iodine.     

Recruitment curve of the soleus H reflex in patients with neurogenic claudication

An abnormal stimulation of the cAMP pathway has been recognized as the primary event in various pathological situations that lead to goitrogenesis or thyroid tumors. Thyroid adenomas are monoclonal neoplasms that become independent of thyroid stimulating hormone (TSH) in their secretory function and growth. Mutated forms of the TSH receptor (TSHR) and the adenylyl cyclase-activating Gs alpha protein, which confer a constitutive activity on these proteins, have been observed in human adenomas. The FRTL-5 rat thyroid cell line is a permanent but untransformed line; the growth of which depends on the presence of TSH, and at least in part, on the stimulation of the cAMP pathway. In order to compare the oncogenic potential of the activated mutant Gs alpha protein and the constitutively activated TSHR, we have transfected FRTL-5 cells with an expression vector bearing either the cDNA of the Gs alpha gene carrying the A201S mutation or the cDNA of the TSH receptor carrying the M453T mutation recently identified in a case of congenital hyperthyroidism. The expression of these two cDNAs was driven by the bovine thyroglobulin gene promoter. We show that, although the expression of both the Gs alpha or TSHR mutant proteins leads to TSH-independent proliferation and to constitutive cAMP accumulation in FRTL-5 cells, only the mutant TSHR is able to induce neoplastic transformation, as demonstrated by growth in semi-solid medium and tumorigenesis in nude mice.     

Inhibition of c-ras reactivates thyroglobulin synthesis in middle-T transformed thyroid cells

Differentiated thyroid cells expressing polyoma Middle-T became transformed and tumorigenic when injected into syngenic animals. The expression of thyroglobulin was greatly reduced and no longer responsive to thyrotropin (TSH) and to cAMP. Inhibition of endogenous c-ras by the expression of two transdominant negative mutant H-ras genes, Asn17 and Leu61-Ser186, reactivated thyroglobulin synthesis. Reactivation of thyroglobulin synthesis by c-ras inhibition was not observed in the absence of TSH. These findings indicate that MT elicits dedifferentiation of thyroid cells by activating endogenous c-ras and that c-ras interferes with TSH or cAMP signaling.     

Structures important in NAD(P)(H) specificity for mammalian retinol and 11-Cis-retinol dehydrogenases

The effects of insulin-like growth factor I (IGF-I) on mitogenesis, epithelial barrier function and transepithelial iodide transport were studied in confluent, polarized monolayers of pig thyrocytes cultured on filter in Transwell bicameral chambers. The growth rate in controls cultured in 1% fetal calf serum was low. Insulin-like growth factor I stimulated dose-dependently the incorporation of [3H]thymidine, maximally at 100 ng/ml, which corresponded to an increase of DNA content by 60% after 6 days. Thyrotropin (1 mU/ml) alone did not stimulate cell multiplication but inhibited partially the stimulatory effect of IGF-I. Insulin-like growth factor I (100 ng/ml) increased within 10 min the transepithelial potential difference, which remained elevated for several days, but did not significantly change the transepithelial resistance. When added together, IGF-I reinforced the effects of TSH on potential difference (increase) and resistance (decrease). A preserved epithelial barrier in IGF-I-treated cultures was confirmed by observing a normal immunolocalization of the tight junction protein ZO-1 and an unchanged ultrastructure of the junctional complex. Insulin-like growth factor I increased the transepithelial flux of 125I- in the basal-to-apical, but not in the opposite, direction. Stimulation of iodide transport by IGF-I was modest after 2 days and pronounced after 6 days. In comparison, TSH-stimulated iodide transport was higher after 2 days but lower after 6 days. Both TSH and IGF-I were strongly synergistic, after 6 days amounting to a 90-fold increase over the control basoapical 125I- transfer. The simultaneous accumulation of 125I- in the cell layer was increased two- to fourfold by IGF-I and/or TSH. In conclusion, IGF-I is able to induce growth in preformed monolayers of pig thyrocytes cultured on permeable filter. During these conditions, the mitogenic effect of IGF-I is partially inhibited by TSH, which has no growth-promoting action on its own. The transepithelial transport of iodide and bulk electrolytes is altered by IGF-I without affecting the epithelial barrier function. Specifically, IGF-I up-regulates the activity of the basolateral iodide pump and increases the iodide permeability of the apical plasma membrane. The action of IGF-I on iodide transport is independent of, although synergistic with, that of TSH. The findings support the notion that IGF-I may be an important regulator of thyroid growth and differentiated functions.     

Thyroid hormone production and its regulation]

Thyroid gland fulfills two functions. On one hand, it synthesizes and builds up stocks of thyroid hormones in thyroglobulin molecules of the colloid in its follicles, such as they can maintain the hormonal secretion during several days and even weeks. To do this, it captures and concentrates plasma iodide through a specific membrane transporter and it oxidizes iodide through the action of thyroperoxidase and H2O2. This makes it able to bind to tyrosine residus of thyroglobulin. Then, the iodotyrosines can form the thyroid hormones (T4 and T3) by a coupling reaction. On the other hand, thyroid secretes the hormones after internalization and proteolysis of thyroglobulin. All the steps of synthesis and secretion are regulated by pituitary TSH, through a negative feed-back action of T4 and T3. Thus, any increase or decrease of circulating thyroid hormones induces the opposite modification of TSH. In addition, an important fraction of plasma and tissue T3 is produced through the extrathyroidal monodeiodination of T4 by enzymes (5' deiodases) which are regulated by the nutritional status and by thyroid hormones.     

Retarded incorporation of [14C]mannose into thyroglobulin of human thyrotoxic thyroid glands

In vitro studies using thyroid slices from human non-toxic goitres and from thyrotoxic glands show retarded incorporation of [14C]mannose into the 19S protein of thyrotoxic glands. This was not found using [14C]galactose with thyrotoxic glands or using either labelled sugar with slices from non-toxic goitres. Experiments with thyroid tissue from rats on a variety of treatment regimes such as iodine supplements, carbimazole alone or with iodine supplements did not show this differential delay of [14C]mannose incorporation. This suggests that there may be some abnormality of carbohydrate incorporation into thyroglobulin in thyrotoxicosis.     

Inhibitory effects of tunicamycin and 2-deoxyglucose on thyroglobulin synthesis

The kinetic incorporation of labelled sugars and amino acids by rat thyroid hemilobes was measured in the presence of 2-deoxyglucose and tunicamycin, inhibitors of the glycosylation of glycoproteins. With either inhibitor the carbohydrate content of exocytosed thyroglobulin was only slightly decreased (less than 20% of control) whereas the rate of exocytosis was strongly inhibited (by 60-80%). As no intracellular accumulation or proteolysis of non-glycosylated molecules was detected, the reduced rate of thyroglobulin release seems essentially due to a decrease in protein synthesis. In a whole cell system (hemilobes), it is impossible to uncouple glycosylation and protein synthesis by incubation with tunicamycin; 50 micrograms/ml tunicamycin for 270 min inhibited total [3H]-glucosamine and 14C-labelled amino acid incorporation by 65% and 33% respectively. This can be contrasted with cell-free incubation of thyroid rough microsomes where glycosylation was blocked by the same tunicamycin concentration (90% inhibition of N-[3H]acetylglucosamine transfer from UDP-N-[3H]acetylglucosamine) whilst ongoing protein synthesis was not significantly modified (less than 4% inhibition). This clearly suggests that, in thyroid follicular cells, a regulatory link exists between the synthesis of the peptide moiety of a glycoprotein and its glycosylation.     

Sphingosine 1-phosphate mobilizes sequestered calcium, activates calcium entry, and stimulates deoxyribonucleic acid synthesis in thyroid FRTL-5 cells

Sphingosine 1-phosphate (SPP) potently mobilizes sequestered calcium and is a mitogen in several cell types. In the present investigation, we have evaluated the effect of SPP on intracellular free calcium concentration ([Ca2+]i) and synthesis of DNA in thyroid FRTL-5 cells. SPP rapidly and transiently mobilized sequestered calcium and stimulated entry of extracellular calcium. The entry of calcium, but not the mobilization, was in part inhibited by pretreatment with pertussis toxin (Ptx), and by activation of protein kinase C. SPP did not stimulate the production of inositol 1,4,5-trisphosphate. SPP stimulated the incorporation of 3H-thymidine in a time- and dose-dependent manner. The effect was not inhibited by Ptx. Furthermore, SPP stimulated the activation of the proto-oncogene c-fos. SPP rapidly tyrosine-phosphorylated an approximately 66 kDa protein. This phosphorylation persisted for at least 1 h. Pretreatment of the cells with genistein abolished the SPP-evoked tyrosine phosphorylation, and attenuated the SPP-evoked increase in [Ca2+]i. Furthermore, the SPP-evoked activation of Na+-H+ exchange was inhibited by genistein. The phosphorylation was not attenuated by pretreatment of the cells with Ptx. SPP per se did not affect cellular cAMP levels but attenuated the TSH-evoked increase in cAMP. As the effect of SPP might be due to activation of phospholipase D, we tested whether phosphatidic acid (PA) mobilized calcium or stimulated the incorporation of 3H-thymidine. PA mobilized sequestered calcium but did not stimulate calcium entry. PA very modestly enhanced the incorporation of 3H-thymidine. Our results suggest, that SPP stimulates DNA synthesis and activates entry of calcium in FRTL-5 cells. The effect on calcium entry appears to be dependent, at least in part, on one or several tyrosine kinases.     

Neonatal transient hypothyroidism: aetiological study. Italian Collaborative Study on Transient Hypothyroidism

AIMS: To define the aetiology of neonatal transient hypothyroidism (NTH) and recommend preventive measures. METHODS: Maternal and perinatal clinical data on the use of antiseptics, drugs, and contrast agents containing iodine were collected from 40 subjects. Thyroid stimulating hormone (TSH), free thyroxine (FT4), thyroxine (T4), thyroglobulin (TG), TSH receptor antibodies, thyroid peroxidase antibodies and urinary iodine were measured in random neonatal samples. In the mothers with known or suspected thyroid disorders, TSH, FT4, TSH receptor antibodies and thyroid peroxidase antibodies were also measured. RESULTS: The NTH aetiology was identified in 85% of cases. More than 50% of the babies with transient hypothyroidism had been exposed to iodine; maternal transfer of antibodies had occurred in a third of them. CONCLUSIONS: It is suggested that the practice of using iodine containing disinfectants should be withdrawn, and chlorhexidine substituted instead; that pregnant women should be advised of the adverse effects of using iodine products; and that thyroid function should be monitored whenever iodine is used.