Exposure of pregnant women to persistent organic pollutants and cord sex hormone levels.

STUDY QUESTION: Is prenatal exposure to persistent organic pollutants (POPs) associated with variations of sex hormone levels in cord blood? SUMMARY ANSWER: Prenatal exposure to a number of POPs is associated with a disruption of hormone levels in cord blood, with sex specificities. WHAT IS KNOWN ALREADY: Epidemiological studies have reported disorders of reproductive health, in relation with POPs exposure during early life and the endocrine disruption properties of these chemicals have been suggested as possible mechanisms. STUDY DESIGN, SIZE, DURATION: A subset of 282 mother-child pairs was selected from the prospective population-based PELAGIE birth cohort (n = 3421, 2002-2006, Brittany, France). Pregnant women were recruited before 19 weeks of gestation and followed until delivery. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sex hormone levels including sex hormone-binding globulin (SHBG), estradiol (E2), total testosterone (T), free testosterone (fT = T/SHBG) and the aromatase index (AI = T/E2) were measured in 282 cord blood samples. Anti-Müllerian hormone (AMH) was measured in male newborns only. Pesticide concentrations of α-endosulfan, ß-hexachlorocyclohexane (ß-HCH), γ-HCH, dieldrin, pp'-dichlorodiphenyldichloroethylene (p,p'-DDE), hexachlorobenzene (HCB), heptachlor epoxide (HCE), as well as PCBs (congeners 153, 187 and the sum of anti-estrogenic PCBs 118, 138, and 170) and decabrominated diphenyl ether (BDE209) were also measured in cord blood. Associations between sex hormones and POPs exposure were explored using multiple linear regressions adjusted for potential confounders. MAIN RESULTS AND THE ROLE OF CHANCE: High PCB levels were associated with an increase of SHBG (P-trend < 0.01) and AMH (P-trend < 0.05) and a decrease of fT (P-trend < 0.05) and AI (P-trend < 0.01). High pesticide levels, particularly α-endosulfan and HCE, were associated with an increase of SHBG (P < 0.05) and E2 (P < 0.01) and a decrease of fT (P < 0.05) and AI (P < 0.01). Several of these associations were stronger, or specific, among male or female newborns. The associations were not altered in the sensitivity analyses. LIMITATIONS, REASONS FOR CAUTION: The study population was of relatively small sample size, and some compounds rarely detected in cord blood. The high level of correlation between POPs makes it difficult to identify the most contributing POPs. Hormone measurements were performed at birth (in cord blood) and may not adequately represent the infant endocrine system. Multiple statistical testing may have led to false-positive associations. WIDER IMPLICATIONS OF THE FINDINGS: Our results are in discordance with those reported in the only published study of the kind but in accordance with studies about prenatal exposure to other endocrine disruptors such as phthalates. These findings may help understanding the pathways involved in adverse reproductive outcomes associated with POPs exposure. STUDY FUNDING/COMPETING INTERESTS: The PELAGIE cohort is funded by Inserm, French Ministry of Health, French Ministry of Labor, InVS, ANR, ANSES, and French Ministry of Ecology. None of the authors has any competing interest to declare.

Anoctamin-1/TMEM16A is the major apical iodide channel of the thyrocyte.

Iodide is captured by thyrocytes through the Na(+)/I(-) symporter (NIS) before being released into the follicular lumen, where it is oxidized and incorporated into thyroglobulin for the production of thyroid hormones. Several reports point to pendrin as a candidate protein for iodide export from thyroid cells into the follicular lumen. Here, we show that a recently discovered Ca(2+)-activated anion channel, TMEM16A or anoctamin-1 (ANO1), also exports iodide from rat thyroid cell lines and from HEK 293T cells expressing human NIS and ANO1. The Ano1 mRNA is expressed in PCCl3 and FRTL-5 rat thyroid cell lines, and this expression is stimulated by thyrotropin (TSH) in rat in vivo, leading to the accumulation of the ANO1 protein at the apical membrane of thyroid follicles. Moreover, ANO1 properties, i.e., activation by intracellular calcium (i.e., by ionomycin or by ATP), low but positive affinity for pertechnetate, and nonrequirement for chloride, better fit with the iodide release characteristics of PCCl3 and FRTL-5 rat thyroid cell lines than the dissimilar properties of pendrin. Most importantly, iodide release by PCCl3 and FRTL-5 cells is efficiently blocked by T16Ainh-A01, an ANO1-specific inhibitor, and upon ANO1 knockdown by RNA interference. Finally, we show that the T16Ainh-A01 inhibitor efficiently blocks ATP-induced iodide efflux from in vitro-cultured human thyrocytes. In conclusion, our data strongly suggest that ANO1 is responsible for most of the iodide efflux across the apical membrane of thyroid cells.

H2O2, signal, substrate, mutagen and chemorepellent from physiology to biochemistry and disease.

The history of the study by our group of the generation, the role and the effects of H2O2 in the thyroid, is summarized. The relations with thyroid diseases are discussed: myxedematous cretinism, thyroiditis, thyroid cancer, congenital hypothyroiddism, are discussed. A new role of H2O2 in the chemorepulsion of bacteria is proposed.

[New assays of TSH-receptor antibodies: analytical and clinical performances in the diagnosis of Graves' disease]. / Nouveaux dosages des anticorps anti-récepteur de la TSH : performances analytiques et cliniques dans le diagnostic de la maladie de Basedow.

Graves' disease autoimmunity is attributable to the presence of serum antibodies (Ab) directed against the TSH receptor (TSHR) measured by a second generation (2G) assay using the human TRAK (hTRAK) with a high sensitivity in the diagnosis of Graves' disease. In this study, we have compared both analytical and clinical performances of hTRAK with those of five new methods using a porcine TSHR: two 2G methods and three assays using the monoclonal M22 directed against the TSHR pocket. We showed a bad reproducibility of these new methods with inter assay CVs higher than 10%. High clinical sensitivity and specificity that appeared similar to those of the hTRAK and next to 100% were observed except for a 2G method that failed to detect five Graves' patients. All these new methods should be avoided since they display a high variability despite their calibration against the same International Standard 90/672. The TRAKh using a human TSHR should be still used for a correct interpretation of results in the follow-up of Graves' disease.

Acrylamide, an in vivo thyroid carcinogenic agent, induces DNA damage in rat thyroid cell lines and primary cultures.

Chronic treatment of rats with acrylamide induces various tumors among which thyroid tumors are the most frequent. The aim of the present study was to develop an in vitro model of acrylamide action on thyroid cells to allow the investigation of the mechanism of this tumorigenic action. The first part of the study considered as targets, characteristics of thyroid metabolism, which could explain the thyroid specificity of acrylamide action: the cAMP mitogenic effect and the important H2O2 generation by thyroid cells. However, acrylamide did not modulate H2O2 or cAMP generation in the thyroid cell models studied. No effect on thyroid cell proliferation was observed in the rat thyroid cell line FRTL5. On the other hand, as shown by the comet assay, acrylamide induced DNA damage, as the positive control H2O2 in the PC Cl3 and FRTL5 rat thyroid cell lines, as well as in thyroid cell primary cultures. The absence of effect of acrylamide on H2AX histone phosphorylation suggests that this effect does not reflect the induction of DNA double strand breaks. DNA damage leads to the generation of mutations. It is proposed that such mutations could play a role in the carcinogenic effect of acrylamide. The mechanism of this effect can now be studied in this in vitro model.

Serum insulin-like growth factor-I measurement in the follow-up of treated acromegaly: comparison of four immunoassays.

BACKGROUND: Measurement of serum insulin-like growth factor I (IGF-I) is currently used for the diagnosis and for monitoring treated acromegaly. In this work, we have studied the performances of four IGF-1 immunoassays in the follow-up of acromegaly. METHODS: The study was carried out on 52 sera from 40 patients with treated acromegaly. Serum IGF-I was measured with radioimmunoassays from Immunotech, DiaSorin and Schering Laboratories and by the chemiluminescent automated immunoassay (Advantage) from Nichols. Normal age- and gender-matched subjects constituted the reference population for two assays. RESULTS: Deming regression and Bland-Altman analyses showed a high correlation with the four methods tested. The use of restricted number of age-adjusted controls with the Immunotech assay, the DiaSorin assay and the Schering assay induced discordance with the Nichols assay in the interpretation of results in treated acromegaly. When normal ranges of the Immunotech assay and the DiaSorin assay were defined from large number of controls, the frequency of normal IGF-I became similar as that obtained with the Nichols assay. CONCLUSION: The four immunoassays display suitable analytical performance for serum IGF-I measurement in the follow-up of acromegaly. For correct interpretation, IGF-I normal ranges should be defined in a large number of age-adjusted healthy subjects.

Ten-year improvement of insulin resistance and growth with recombinant human insulin-like growth factor 1 in a patient with insulin receptor mutations resulting in leprechaunism.

AIM: Leprechaunism, a rare genetic disease resulting from mutations in two alleles of the insulin receptor gene, is characterized by severe insulin resistance, retarded growth and, usually, premature death. The ability of treatment with recombinant human insulin-like growth factor 1 (rhIGF1) to improve metabolic and clinical parameters in the long-term is still controversial. METHODS: Mutations were looked for in the insulin receptor gene of a four-month-old female baby with leprechaunism. The patient's skin fibroblasts were analyzed for response to insulin and IGF1. At the clinical level, the very long-term effects of treatment with rhIGF1/rhIGFBP3 were evaluated by clinical and metabolic parameters. RESULTS: The patient's diagnosis was based on compound heterozygous mutations in two alleles of the insulin receptor gene, thus confirming leprechaunism. Cultured fibroblasts showed a decreased number of insulin receptors and were insulin-resistant. However, IGF1 was able to stimulate IGF1 receptor signalling, suggesting possible activation of a salvage pathway. Treatment with IGF1/IGFBP3 for 8.7 years, then IGF1 for 2 years, resulted in normalization of circulating levels of IGF1 and IGFBP3. Large daily variations in glycaemia and insulinaemia persisted, but mean glycaemia decreased. Regarding growth, the patient's BMI Z score normalized and length/height score improved. Our patient presented normal neurological development and academic achievement. The treatment was free of adverse effects. CONCLUSION: Our results provide evidence that rhIGF1 with and without rhIGFBP3 can prevent fatal outcomes, and improve growth and metabolic parameters, for more than 10 years in a patient with leprechaunism. Long-term rhIGF1 for severe insulin resistance syndrome should be considered.

Anion selectivity by the sodium iodide symporter.

The iodide transporter of the thyroid (NIS) has been cloned by the group of Carrasco. The NIS-mediated transport was studied by electrophysiological methods in NIS-expressing Xenopus oocytes. Using this method, the anion selectivity of NIS was different from that previously reported for thyroid cells, whereas perchlorate and perrhenate were found not transported. In this study we compared the properties of human NIS, stably transfected in COS-7 cells to those of the transport in a thyroid cell line, the FRTL5 cells, by measuring the transport directly. We measured the uptake of (125)I(-), (186)ReO(4)(-), and (99m)TcO(4)(-) and studied the effect on it of known competing anions, i.e. ClO(4)(-), SCN(-), ClO(3)(-), ReO(4)(-), and Br(-). We conclude that the properties of the NIS transporter account by themselves for the properties of the thyroid iodide transporter as described previously in thyroid slices. The order of affinity was: ClO(4)(-) > ReO(4)(-) > I(-) >/= SCN(-) > ClO(3)(-) > Br(-). NIS is also inhibited by dysidenin (as in dog thyroid).

Sphingolipid-cholesterol domains (lipid rafts) in normal human and dog thyroid follicular cells are not involved in thyrotropin receptor signaling.

Partition of signaling molecules in sphingolipid-cholesterol-enriched membrane domains, among which are the caveolae, may contribute to signal transduction efficiency. In normal thyroid, nothing is known about a putative TSH/cAMP cascade compartmentation in caveolae or other sphingolipid-cholesterol-enriched membrane domains. In this study we show for the first time that caveolae are present in the apical membrane of dog and human thyrocytes: caveolin-1 mRNA presence is demonstrated by Northern blotting in primary cultures and that of the caveolin-1 protein by immunohistochemistry performed on human thyroid tissue. The TSH receptor located in the basal membrane can therefore not be located in caveolae. We demonstrate for the first time by biochemical methods the existence of sphingolipid-cholesterol-enriched domains in human and dog thyroid follicular cells that contain caveolin, flotillin-2, and the insulin receptor. We assessed a possible sphingolipid-cholesterol-enriched domains compartmentation of the TSH receptor and the alpha- subunit of the heterotrimeric G(s) and G(q) proteins using two approaches: Western blotting on detergent-resistant membranes isolated from thyrocytes in primary cultures and the influence of 10 mm methyl-beta-cyclodextrin, a cholesterol chelator, on basal and stimulated cAMP accumulation in intact thyrocytes. The results from both types of experiments strongly suggest that the TSH/cAMP cascade in thyroid cells is not associated with sphingolipid-cholesterol-enriched membrane domains.

Kinetics of thyrotropin-stimulating hormone (TSH) and thyroid-stimulating antibody binding and action on the TSH receptor in intact TSH receptor-expressing CHO cells.

The kinetics of TSH binding and the effects of TSH and thyroid-stimulating antibody (TSAb) on cAMP accumulation have been measured in TSH receptor-expressing CHO cells (CHO-TSHR cells). The parallel kinetics of TSH binding to its receptor and of cell cAMP concentration after the addition and withdrawal of TSH show that in the case of this receptor, signal generation and concentration are at all times proportional to occupancy. In physiological ionic medium, TSAb, but not TSH, action is slowed and in some cases almost nonexistent. The kinetics of cAMP disappearance after washout of TSAb is also slower. cAMP accumulation is faster for Fabs than for the TSAb from which they derive. Analysis of the data suggest that 1) serum TSAb are oligoclonal antibodies sets, at low concentrations, with a high affinity for the TSH receptor; 2) ionic interactions are involved in the action of TSAb on the TSH receptor; and 3) TSAb activation of the TSH receptor is at least a two-step process. Among others, a possible explanation is that the full activation of the receptor requires the binding of two or more different antibody molecules on different sites of the same TSH receptor. This analysis provides a benchmark for studies of experimentally induced monoclonal antibodies activating the TSH receptor.

Modulation of the functional properties of human thyrocytes in monolayer or follicle culture: effects of some anaesthetic drugs.

It is well known that some volatile anaesthetic drugs, such as halothane and isoflurane, alter the functions of the human thyroid gland, but the action of other anaesthetic drugs, such as thiopental, midazolam and ketamine, on thyroid function is still unknown. We have investigated the effects of these three drugs on the functional properties of human thyrocytes cultured in monolayers or follicles and stimulated by TSH. Thiopental, midazolam and ketamine induced total suppression or a partial reduction, depending on the dose administered, of cyclic AMP (cAMP), follicular thyroglobulin (Tg) and free tri-iodothyronine (FT3) production. In contrast, free thyroxine levels increased in the medium of thyrocytes cultured as follicles. Small doses of the drugs did not affect thyrocyte production. The inhibiting effect of thiopental, midazolam and ketamine on Tg and FT3 production seems to result from the inhibition of cAMP production and 5'-deiodinase.

Effects of cisplatin on human thyrocytes in monolayer or follicle culture.

Cis-diamminedichloroplatinum (II) (cisplatin) is a widely used anticancer drug which induces many side-effects, but its action on the thyroid gland is still unknown. We have investigated the effects of this drug on human thyrocytes cultured in monolayers or in follicles and stimulated with 200 microU TSH/ml. After 72 h in culture, different concentrations of cisplatin (15, 30 and 75 microM) caused partial or total inhibition of cyclic AMP (cAMP), thyroglobulin (Tg) and tri-iodothyronine (T3) production, whereas thyroxine levels increased in the medium of thyrocytes cultured as follicles. Small doses of the drug did not affect thyrocyte production. Decreases in neutral-red uptake by thyroid cells and in intracellular lactate dehydrogenase, alpha-hydroxybutyryldehydrogenase and creatine phosphokinase activities were induced by 30 and 75 microM cisplatin. These data show that high concentrations of cisplatin had a cytotoxic effect on thyrocytes. Cisplatin also induced inhibition of the production of cAMP, Tg and T3.

Expression of membrane and soluble intercellular adhesion molecule-1 in Graves' disease.

We have investigated the in vitro expression of membrane and soluble intercellular adhesion molecule-1 (ICAM-1) by human thyroid cells from 20 patients with Graves' disease and 5 normal subjects. Membrane ICAM-1 was not detected by flow cytometry analysis in non-cultured thyrocytes from either normal or Graves' disease tissues. It appeared on thyroid cells after a 24-h culture in monolayers and showed a regular dose-dependent increase. The same results were obtained with soluble ICAM-1 (sICAM-1) in culture media from cells cultured in monolayers, vesicles or follicles. No change was obtained with different concentrations of fetal calf serum added to the media. Coculture of Graves' disease thyrocytes with autologous peripheral blood lymphocytes (PBL) or intrathyroidal lymphocytes (ITL) enhanced the expression of both membrane and sICAM-1 whatever the culture model. When normal thyrocytes were cocultured with PBL, sICAM-1 increased but with ITL sICAM-1 remained unchanged. High concentrations of gamma interferon induced an increase of both membrane and sICAM-1 in the three culture models. However the increases were greater with vesicles and follicles. Only sICAM-1 levels were raised with 0.1, 1 and 10 microM retinoic acid. These results suggest that ICAM-1 appears in culture, possibly due to mechanical effects such as adherence to plates and cell-to-cell contacts. Moreover, its expression is modulated by several factors such as cytokines or retinoic acid. Further investigations are needed to establish whether ICAM-1 is really involved in the pathogenesis of Graves' disease.

Effect of lymphocytes on hormonal secretion by autologous thyrocytes cultured in monolayers.

We studied the lymphocyte-induced alterations in hormonal metabolism and the production of tumour necrosis factor alpha (TNF-alpha) during coculture of thyrocytes and autologous lymphocytes from 20 patients with Graves' disease and from five normal subjects. Thyroglobulin (Tg) mRNA was assessed by slot-blot analysis under TSH stimulation. Tg, tri-iodothyronine (T3) and cAMP secretion in the presence of TSH were measured by RIA after 3 or 5 days of coculture. TNF-alpha levels produced after 5 days incubation were also assayed in lymphocyte culture and coculture media. Lymphocytes isolated from peripheral blood (PBLs) altered the production of Tg, T3 and cAMP in autologous thyrocytes. Intrathyroidal lymphocytes (ITLs) decreased Tg and cAMP secretion but had no effect on T3 secretion. The reductions in Tg and cAMP levels obtained with mechanically isolated ITLs (M-ITLs) were generally higher than those obtained with ITLs isolated by dispase (D-ITLs). No difference was seen between Graves' disease and normal cocultures. PBLs secreted large concentrations of TNF-alpha, larger than those obtained with M-ITLs whereas D-ITLs produced low amounts of this cytokine. In coculture, TNF-alpha levels were lower than those observed in lymphocyte culture. Significant correlations were obtained between TNF-alpha levels and the decrease in Tg, T3 and cAMP concentrations. The percentage of T lymphocytes was higher in PBLs and D-ITLs than in M-ITLs. B lymphocytes levels were higher in ITLs, especially M-ITLs, than in PBLs. TNF-alpha production by B lymphocytes was maximal in M-ITLs. In conclusion, lymphocytes induced a decrease in hormonal thyroid metabolism when cocultured with autologous thyrocytes. These perturbations may be attributed, at least partly, to TNF-alpha secreted by lymphocytes. TNF-alpha interacts via the adenylate cyclase pathway of TSH signal transduction.

Hormonal study of a human mixed follicular and medullary thyroid carcinoma.

We studied the hormonal secretion of a human mixed follicular and medullary carcinoma. Thyroglobulin (Tg) secretion, especially by large cells and sometimes by small ones, was visualized with immunoenzymatic staining. Calcitonin (CT) was produced by small spindle-shaped cells. Moreover, immunofluorescence double staining performed on the resected thyroid tissue showed the secretion of both Tg and CT in a small number of cells. The cells lost their hormonal secretion after 2 months of culture. Hormonal secretion was modulated by different additives in the medium. Tg secretion was induced when TSH was added to the culture medium; the maximal effect was produced with the addition of 1 mU TSH/ml and 1 microM cortisol, which potentiated the effect of TSH on Tg production. A durable Tg secretion was obtained by embedding the cells in Engelbretch-Hohn-Swarn (EHS) tumour matrix. The CT production was reinduced by the addition of 4 mM Ca2+, 1 microM glucagon and 1 microM cortisol to the culture medium. These findings show that different cells are found in a mixed follicular and medullary carcinoma, some of which can secrete both CT and Tg. They can remain differentiated for a long period after being embedded in EHS tumour matrix with Ca2+ and hormonal components.

Action of peripheral or intrathyroidal lymphocytes on autologous thyrocytes cultured in follicles in collagen gel.

We have studied the action of peripheral blood lymphocytes (PBLs) and intrathyroidal lymphocytes (ITLs) on the biochemical and hormonal metabolism of autologous thyrocytes cultured in follicles in a collagen gel. The production of tumour necrosis factor alpha (TNF-alpha) in culture was also measured. Thyroid tissues and lymphocytes were obtained from ten patients with Graves' disease and from five control subjects. Lymphocyte-induced cytotoxicity was evaluated in autologous thyrocytes cultured in a collagen gel by several tests; neutral red uptake, lactate dehydrogenase activity and glutathione level. Hormonal metabolism was assessed by evaluating tri-iodothyronine (T3) and total cAMP production under TSH stimulation. TNF-alpha levels were measured in supernatants after 5 days of coculture. PBLs altered biochemical metabolism, T3 synthesis and cAMP production in autologous thyroid follicles. These inhibitions were greater than those obtained with ITLs. No difference was seen between cells obtained from patients with Graves' disease and those from normal subjects. TNF-alpha levels secreted by PBLs were higher than those secreted by ITLs. The concentrations of this cytokine decreased in coculture. Significant correlations were observed between the decrease in biochemical and hormonal parameters and TNF-alpha levels. Exogenous TNF-alpha and high doses of interferon gamma inhibited follicle metabolism, especially hormone secretion. In conclusion, thyrocytes cultured in follicles provide a more sensitive model than monolayer cultures for analysis of lymphocyte-induced interactions. Lymphocytes gradually inhibit the biochemical and hormonal metabolism of autologous thyroid follicles depending on the isolation method. These alterations may be particularly attributed to TNF-alpha secreted by lymphocytes. The cytokine-induced inhibition of thyroid hormonal function apparently involves the adenylate cyclase system.

Effect of amiodarone and propranolol on the functional properties of human thyroid follicles cultured in collagen gel.

Amiodarone and propranolol have been known to inhibit the peripheral conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) but their direct effect on the thyroid gland is not understood. We therefore investigated the action of the two drugs on the functional properties of human thyroid follicles embedded in collagen gel. Amiodarone and propranolol induced a dose-dependent reduction or blocking of the follicular production of cyclic AMP (cAMP), thyroglobulin (Tg) and free triiodothyronine (FT3) under 200 microU/ml medium. Small drug doses and the drug solvents did not affect the follicular secretion. The inhibiting effect of amiodarone and propranolol on follicular production of cAMP, Tg and FT3 appears to result from several factors: (1) inhibition of thyroid 5'-deiodinase; (2) amiodarone high iodine content; (3) a quinidine-like effect of propranolol involving a membrane-stabilizing mechanism.

Functional properties of human thyroid follicles cultured within collagen gel.

Cultures of human thyroid follicles embedded in collagen gel were performed to investigate certain functional properties under bovine thyrotropin (TSH) stimulation. Follicles obtained from normal glands responded to increasing concentrations of TSH administered on day 4 in culture and for 3 days by increased amounts of cyclic AMP (cAMP), thyroglobulin (Tg) and triiodothyronine (T3) and by decreased levels of thyroxine (T4). Effect was maximal at 2000 microU/ml TSH (cAMP) or 200 microU/ml (Tg, T3, T4). When methimazole or propylthiouracil (PTU) were added, the T3 levels decreased. Follicle lumens contained a periodic acid-Schiff substance which was identified by immunoreaction as Tg. Thyroid follicles obtained from Graves' disease glands gave modified results with an earlier and intensified T3 response and no increase in Tg. These data show that (1) Tg and T3 are secretory products of functional follicles giving a cAMP-mediated response to TSH. (2) The detected T3 also derives from T4 5'-deiodination inhibited by PTU. (3) Intensified T3 response in Graves' follicles is probably due to enhanced conversion of T4 to T3.

Long-term culture of human pancreatic islets in an extracellular matrix: morphological and metabolic effects.

In this experiment, various conditions for embedding cultures of human pancreatic islets in type I collagen gel were studied in an attempt to maintain the highly differentiated functions of islet cells and particularly insulin secretion over a long period of time. The islets isolated by a collagenase digestion technique were plated either on or within the collagen gel and refed with either Eagle's minimum essential medium (5.5 mM D-glucose) or RPMI 1640 medium (11 mM D-glucose) supplemented with 10% FCS and antibiotics. The comparison between the two culture media showed that embedded islets cultured in RPMI had a higher basal insulin secretion rate, survived longer than their MEM counterparts, but exhibited impaired response to an acute glucose test contrasting thus with islets cultured in MEM. The secretory behaviour of islets was also related to the different morphological modifications occurring during culture. Islets directly embedded within the collagen gel more or less maintained their spherical structure and highest secretory capacities. When overlaid with a second layer of collagen, well established monolayers of human islet cells grown on collagen underwent a gradual and complete reorganization into a three-dimensional islet-like structure with a striking reinforcement of their secretory activity. Both cultures were able to survive more than 8 weeks, thus proving the usefulness of such a new model for long-term culture. In contrast, standard cultures on culture treated plastic dishes on which islets cells rapidly established wide monolayers, exhibited a rapid and definitive decline in insulin secretion with a survival not exceeding 14 days. In the light of these different culture conditions, possible mechanisms responsible for disturbance of hormonal release and their implications for in-vitro study of isolated islets functions are discussed. In conclusion, this work is a new example of the permissive effects of collagen matrices on the establishment or maintenance of tissue-like structures in vitro, suggesting the definition of a new model for the study of human pancreatic islets in long-term culture.

Specific activation of the thyrotropin receptor by trypsin.

The identification of 16 different activating mutations in the TSH receptor, found in patients suffering from toxic autonomous adenomas or congenital hyperthyroidism, leads to the concept that this receptor is in a constrained conformation in its wild-type form. We used mild trypsin treatment of CHO-K1 cells or COS-7 cells, stably or transiently transfected with the human TSH receptor, respectively, and measured its consequences on the TSH receptor coupled cascades, i.e. cyclic AMP and inositol-phosphates accumulation. A 2-min, 0.01% trypsin treatment increased stably cyclic AMP but not inositol-phosphates formation. This was not observed after chymotrypsin, thrombin and endoproteinase glu C treatment. The TSH action on cyclic AMP was decreased by only 25%. The effect was also observed in cells expressing the dog TSH receptor. It was not observed in MSH receptor, LH receptor expressing or mock transfected cells (vector alone). It is therefore specific for the TSH receptor, for its action on the Gs/adenylate cyclase cascade, and for the proteolytic cleavage caused by trypsin. Using monoclonal (A. Johnstone and P. Shepherd, personal communication) and polyclonal antibodies directed against the extracellular domain of the TSH receptor, it was shown that treatment by trypsin removes or destroys a VFFEEQ epitope (residues 354-359) from the receptor. The effect mimics the action of TSH as it activates Gs alpha and enhances the action of forskolin. It is not reversible in 1 h. The results support the concept that activation of the receptor (by hormone, autoantibodies, mutations or mild proteolysis) might involve the relief of a built-in negative constrain. They suggest that the C-terminal portion of the large extracellular domain plays a role in the maintenance of this constrain.