Expression of integrins of the beta1 family in thyroid cells from patients with Graves' disease in vivo and in vitro.

The expression of the beta1 family of integrins was determined in thyroid follicular cells from patients with Graves' disease (GD). Integrin expression was quantitated by flow fluorocytometry of single cell suspensions with antibodies against the common beta1 chain and the alpha1-alpha6 subunits. Results indicated that also in thyroid glands of GD, as previously observed in nodular goiters, two follicular cell populations with different patterns of beta1 integrin expression coexist (VLAalpha3beta1 and VLAalpha1,3,5,6beta1). The VLAalpha1,3,5,6beta1 thyrocyte population in GD was more abundant than in nodular goiters, ranging from 40 to 70% of the total follicular cells and the overall expression of the beta1 integrins was a two-fold higher. In thyrocytes from patients with GD cultured in vitro, alpha3 and alpha2 expression was regulated by cell-to-cell contact as previously described in normal thyroid cells, while the expression of alpha1, alpha5 and alpha6 was quickly lost during the culture. Our data suggest that the integrin profile of the VLAalpha1,3,5,6beta1 thyrocyte population in GD is induced by micro-environmental conditions rather than being the expression of a constitutive phenotype.

Laminin receptors in differentiated thyroid tumors: restricted expression of the 67-kilodalton laminin receptor in follicular carcinoma cells.

The expression of integrin laminin receptors was investigated in normal thyroid primary cultures; immortalized normal thyroid cells (TAD-2); papillary (NPA), follicular (WRO), and anaplastic (ARO) thyroid tumor cell lines; seven thyroid tumors (four papillary and three follicular carcinomas); and normal thyroid glands. The expression of alpha1beta1, alpha2beta1, alpha3beta1, alpha6beta1, and alpha6beta4 was found in all tumor specimens and in tumor cell lines, whereas normal thyroid cells and TAD-2 cells lacked the expression of alpha6beta4. Despite the presence of several integrin laminin receptors, adhesion of TAD-2, NPA, and ARO cells to immobilized laminin-1 was poor, whereas WRO cells and follicular carcinoma-derived cells displayed a strong adhesion. Indeed, WRO and follicular carcinoma-derived cells showed expression of a nonintegrin laminin receptor, the 67-kDa high affinity laminin receptor (67LR). TAD-2, NPA, and ARO cells as well as nodular goiter, toxic adenoma, follicular adenoma, and papillary carcinoma-derived cells did not express the 67LR. Adhesion of WRO and follicular carcinoma-derived cells to laminin-1 was specifically inhibited by a recombinant polypeptide containing laminin-binding domains of 67LR, demonstrating that this receptor confers to follicular carcinoma cells attachment capacity to laminin. Moreover, tissue specimens from follicular carcinomas expressed the 67LR, whereas follicular adenomas and normal thyroid tissues were negative. In thyroid tumors, integrin receptors, although abundant, participate weakly in adhesion to laminin. The expression in follicular carcinoma cells of a functional, high affinity 67LR together with nonfunctional integrin LM receptors could be responsible for the tendency of follicular carcinoma cells to metastasize by mediating stable contacts with basal membranes.

Intercellular adhesion molecule-1 is upregulated via the protein kinase C pathway in human thyroid carcinoma cell lines.

Nonantigen specific adhesion systems lymphocyte function-associated antigen 1/intercellular adhesion molecule (LFA-1/ICAM-1) and cluster designation 2/lymphocyte function-associated antigen 3 (CD2/LFA-3) are considered a crucial step in immune-mediated cell-cell adhesion reactions. In particular, the LFA-1/ICAM-1 system is deeply involved in major histocompatibility system (MHC)-restricted and non-MHC-restricted cellular cytotoxicity of effector cells against cancer tissues. We have investigated in human thyroid carcinoma cell lines the role of the protein kinase C (PKC) pathway on ICAM-1 expression. Incubation with tissue plasminogen activator (TPA), an agonist of PKC, of two papillary (NPA and TPC-1) and one anaplastic (ARO) carcinoma cell lines induced an ICAM-1 upregulation of both protein and mRNA production. This phenomenon was dependent on RNA and protein synthesis and was inhibited by PKC antagonists such as staurosporine and H-7. A parallel increase in the soluble form of ICAM-1 followed the upregulation of cellular ICAM-1 levels induced by TPA. In conclusion, the PKC pathway is involved in the regulation of ICAM-1 expression in human thyroid carcinoma cell lines. Further studies are necessary to clarify the effects of the PKC pathway on the diffusion of thyroid tumors.

Retinoic acid induces intercellular adhesion molecule-1 hyperexpression in human thyroid carcinoma cell lines.

The expression of intercellular adhesion molecule-1 (ICAM-1) in tumoral tissues may promote their interaction with the immune system and cytotoxic effect on tumoral cells. This observation led to the investigation of ICAM-1 expression and modulation in different tumoral cell systems in vitro. Recently, retinoic acid-responsive elements have been found in the 5'-regulatory region of the human ICAM-1 gene. In the present study, we investigated, by flow cytometry, the effect of retinoic acid on the surface expression of ICAM-1 in human thyroid carcinoma cell lines. Two papillary (NPA and TPC-1), one follicular (WRO), one anaplastic (ARO) and one immortalized fetal (TAD-2) cell line have been studied. All of them produced constitutively ICAM-1; its surface expression and specific messenger ribonucleic acid (mRNA) levels were increased significantly by retinoic acid in all except the WRO cell line. ICAM-1 hyperexpression by retinoic acid was time dependent, reversible, and dependent on mRNA and protein synthesis. Furthermore, cytokines, such as interferon-gamma and tumor necrosis factor-alpha, both individually and, to a greater extent, in combination with retinoic acid, increased ICAM-1 surface expression and its mRNA levels. In conclusion, retinoic acid is able to induce ICAM-1 up-regulation via mRNA accumulation in human thyroid carcinoma cell lines.

Graves' disease thyroid tissue transplants in scid mice: persistent selectivity in hTcR Va gene family use.

We have analyzed the human T-cell receptor (hTcR) V alpha gene repertoire in thyroid tissue transplants of a patient with hyperthyroid Graves' disease. Blocks of thyroid tissue were transplanted subcutaneously into 10 mice with severe immunodeficiency (scid) and 4 weeks later 5 of the mice were injected intraperitoneally with autologous peripheral blood mononuclear cells (PBMC) (10(7) cells per mouse). After a further 3 weeks, mice were sacrificed and total cellular RNA and cDNA prepared from each of the explants. We used specific olingonucleotides in polymerase chain reactions (PCR) to amplify 18 different human hTcR V alpha gene families and the identity of the PCR fragments was confirmed by Southern blot analysis. Different samples of the donor thyroid tissue consistently expressed 9-10 of the 18 hTcR V alpha gene families screened (V alpha 1-7, 11, 12 & 15). A more marked bias in hTcR V gene family use was seen in each of the explants with a mean of only 2.8 V alpha gene families detected. After 7 weeks of transplantation, the thyroid explants largely reflected some of the same genes seen in the hTcR V gene repertoire of the donor tissue with particularly pronounced expression of V alpha 2 and V alpha 3 gene families. The transplantation of PBMC into the scid mice showed evidence for their accumulation within the transplanted thyroid tissues as judged by the appearance of additional hTcR V gene families expressed in these samples although the specificity of such accumulation remains unclear.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of mild-to-moderate hyperglycaemia per se on glucose production and uptake in the elderly.

OBJECTIVES: In order to better understand the mechanisms responsible for the diminished glucose tolerance that occurs in the elderly, the present study aimed at investigating the effect of mild hyperglycaemia on glucose production and uptake in a group of aged subjects. For comparison, a group of young subjects was simultaneously investigated. METHODS: Seven aged (71.8 +/- 2.3 yrs) and seven young (25.5 +/- 1.7 yrs) healthy non-obese subjects underwent two hyperglycaemic glucose-clamps having as targets plasma glucose levels 7.5 and 10.0 mmol/L. Contemporary infusion of D-[3-3H]-glucose allowed determination of glucose turnover parameters in basal conditions and during the clamps. Endogenous pancreatic secretion was inhibited by somatostatin (8.3 micrograms/min) while glucagon (67 ng/min) and insulin (0.15 mU/kg/min) were replaced by exogenous infusions. RESULTS: In basal conditions, glucose uptake (12.9 +/- 0.5 vs 14.4 +/- 0.4 mumol/kg/min; p < 0.05) and glucose metabolic clearance rate (2.58 +/- 0.15 vs 3.35 +/- 0.10 ml/kg/min; p < 0.01) were lower in elderly vs young subjects. In the hyperglycaemic glucose-clamps, we observed, in the elderly subjects, the persistence of a greater glucose production during mild (7.5 mmol/L) (11.6 +/- 0.4 vs 9.7 +/- 0.2 mumol/kg/min; p < 0.005) but not moderate (10 mmol/L) (3.5 +/- 0.1 vs 3.4 +/- 0.1 mumol/kg/min; NS) hyperglycaemia. In contrast, glucose-induced glucose uptake and glucose metabolic clearance rate were similarly affected by glucose infusions in both groups of subjects. Moreover, in elderly but not in young subjects, basal glucose disappearance rate was significantly negatively correlated with fasting plasma glucose levels (r = -0.84; p < 0.01). CONCLUSIONS: In the basal state, glucose uptake and glucose metabolic clearance rate are slightly impaired in elderly, compared to young subjects. Furthermore, in the elderly, endogenous glucose production is less suppressed by mild hyperglycaemia i.e. 7.5 mmol/L, than it is in young people. Such impairment in the inhibition of endogenous glucose production is not seen when blood glucose attains 10 mmol/L. We suggest that impairment in glucose tolerance in the elderly results from both reduced glucose uptake (in basal conditions) and excessive glucose production (at mild hyperglycaemic levels).

Study of an endemic goiter area in northern Campania.

The purpose of this paper was the determination, in the endemic goiter area of Teano (Caserta, Italy), of: i) The goiter prevalence in a group of 920 patients who attended the Outpatient Endocrinology Department; ii) The urinary iodine excretion in 150 adults (20-73-year-old) and 502 children (10-16-year-old; iii) The thyroid size in the 502 children; iv) The environmental iodine levels. Out of 920 patients a total of 750 (81.5%) goiters were detected. Out of these 750 cases, 415 (55.3%) were of grades 1b and 2, 335 (44.7%) of grades 3 and 4. A statistically significant association between goiter size and age was found (p less than 0.05). Laboratory data were entirely available for 506 goitrous patients. Serum TG levels was increased with goiter size and age, whereas there was a progressive decrease in mean serum TSH levels with increasing goiter size and age. The screening program performed on 502 schoolchildren aged 10 to 16 yr found a 68.3% prevalence of grade 1 goiter and a mean urinary ratio iodine/creatinine of 52 +/- 32 (SD) micrograms/g. A mean urinary ratio iodine/creatinine of 60 +/- 27 (SD) micrograms/g was reported in a sample of 150 adult inhabitants. Iodine measurements in water supplies showed levels equal to or less than 1 microgram/l. The area investigated can be identified as a moderate iodine deficient area and classified as grade 1-2 according to the Pan American Health Organization criteria.

Insulin resistance and hyperinsulinemia in patients with chronic congestive heart failure.

Congestive heart failure is a condition associated with increased plasma norepinephrine levels. Moreover, norepinephrine has been recently demonstrated to affect glucose homeostasis by decreasing insulin sensitivity. In the present study, eight patients suffering from chronic congestive heart failure and 10 healthy age- and body mass index-matched subjected were submitted to both an oral glucose tolerance test (OGTT; 75 g) and a euglycemic hyperinsulinemic glucose clamp. During the 360 minutes of the glucose clamp, insulin was infused at three different rates (25, 50, and 100 mU/kg/h), while D-3H glucose infusion allowed determination of glucose turnover. In basal conditions, patients versus controls had similar plasma glucose (5.2 +/- 0.1 v 4.9 +/- 0.2 mmol/L,P = NS), but higher plasma insulin (125.7 +/- 9.2 v 35.7 +/- 3.3 pmol/L,P less than .01), norepinephrine (5.39 +/- 0.13 v 1.47 +/- 0.22 nmol/L,P less than .001), and free fatty acid (FFA) (927 +/- 79 v 792 +/- 88 mumol/L,P less than .05) levels. In patients, basal plasma norepinephrine correlated with FFA levels (r = .65, P less than .025). After loading glucose, plasma glucose and insulin levels were still significantly higher in patients than controls. Euglycemic hyperinsulinemic glucose clamp produced a lower insulin-mediated inhibition of endogenous (hepatic) glucose production (HGP) and a greater increase in both glucose disappearance rate (Rd) and glucose metabolic clearance rate (gMCR) in patients than in controls during the first two insulin infusion rates (25 and 50 mU/kg/h). By contrast, these differences disappeared during the highest insulin infusion rate (100 mU/kg/h). Insulin-mediated decrease in plasma FFA levels was also lower in patients than controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Simvastatin reduces plasma lipid levels and improves insulin action in elderly, non-insulin dependent diabetics.

Twelve elderly non-insulin dependent diabetic patients took part in a double-blind, cross-over, randomized study comparing simvastatin 30 mg/day and placebo. Each treatment period lasted 3 weeks and was separated by a 3 week wash-out period. At the end of each treatment period all subjects underwent in randomized order an oral glucose tolerance test (OGTT; 75 g) and an euglycaemic hyperinsulinaemic (50 mU/kg.h) glucose clamp. Simvastatin compared to placebo significantly reduced plasma total cholesterol (7.9 vs 5.3 mmol.l-1), LDL-cholesterol (7.2 vs 4.3 mmol.l-1), triglycerides (2.9 vs 2.1 mmol.l-1), free fatty acids (1106 vs 818 mmol-1) and glucose (7.4 vs 6.6 mmol.l-1) levels. After simvastatin, and in the last 60 min of the glucose clamp, there was an improvement in the action of insulin as demonstrated by stronger inhibition of hepatic glucose output (2.7 vs 5.2 mumol.kg-1.min-1) and stimulation both of the glucose disappearance rate (26.3 vs 19.5 mumol.kg-1.min-1) and glucose metabolic clearance rate (4.3 vs 3.6 ml.kg-1.min-1). The changes in glucose turnover parameters were significantly correlated with basal plasma free fatty acids and were independent of plasma glucoregulatory hormones. In conclusion, simvastatin seems to exert beneficial effects both on lipid and glucose metabolism.

Effects of oxytocin upon the endocrine pancreas secretion and glucose turnover in normal man.

In normal man oxytocin infusion under basal conditions and at pharmacological doses evoked a rapid surge in plasma glucose and glucagon levels followed by a later increase in plasma insulin levels. Simultaneous [D-3H]glucose infusion indicated that oxytocin also produced a prompt and significant increase in hepatic glucose output with a secondary increase in glucose disappearance rate. Eight healthy volunteers were studied during euglycemic glucose clamp and simultaneous [D-3H]glucose infusion, during suppression of endogenous pancreatic secretion by cyclic somatostatin (250 micrograms/h) and during exogenous glucagon (67 ng/min) and insulin (0.15 mU.kg-1.min-1 from 0 to 120 min and 0.40 mU.kg-1.min-1 from 121 to 240 min) replacement. During the first 60 min oxytocin (0.2 U/min) evoked a transient but significant increase in plasma glucose levels and hepatic glucose output with a simultaneous suppression of the glucose infusion rate. No difference in glucose disappearance and metabolic clearance rates were recorded throughout the clamp irrespective of whether oxytocin was infused or not. So we conclude that oxytocin exerts a hyperglycemic effect through an A-cell stimulation and a glycogenolytic action.

Impaired insulin-mediated erythrocyte magnesium accumulation is correlated to impaired insulin-mediated glucose diposal in aged non-diabetic obese patients.

Basal erythrocyte magnesium levels were significantly lower in obese than lean subjects. In vitro incubation in the presence of 100 mU/l insulin significantly increased magnesium erythrocyte levels in both groups of subjects. However, even in the presence of 100 mU/l, the erythrocyte magnesium content of obese patients was lower than that of control subjects. The in vitro dose-response curve of the effect of insulin on magnesium erythrocyte accumulation was shifted to the right when the red cells of obese were used, with a highly significant reduction of the maximal effect. Such reduction of the maximal effect of insulin suggests that the impairment of insulin-induced erythrocyte magnesium accumulation observed in obese patients results essentially from a post-receptor defect. In obese patients, net increase in erythrocyte magnesium levels (calculated as the difference between basal and 100 mU/l insulin-induced erythrocyte magnesium levels) was negatively correlated with basal plasma insulin levels (r = 0.79 p less than 0.01), and with body mass index (r = 0.81 p less than 0.01) while it was positively correlated with the glucose disappearance rate after glucose load (r = 0.67 p less than 0.05) and glucose metabolic clearance rate (r = 0.71 p less than 0.01). These results demonstrate that insulin-induced erythrocyte magnesium accumulation is impaired in patients with obesity and that such defect is correlated to impaired -- mediated glucosal disposal in the patients.

Insulin resistance as cause of increased blood pressure in the elderly: effects on intracellular ion contents.

Previous reports have evidenced a strong relationship between high plasma insulin levels and blood pressure in diabetic and obese subjects but not in the elderly. During aging many patho-physiological changes in cardiovascular functions and autonomic nervous system occur, so that aging per se might be a cause of a 'physiological' increase in blood pressure. Nevertheless, an insulin resistance also develops during aging. The present study investigates the possible role of age-dependent insulin resistance in the genesis of increased blood pressure. Our data show that insulin resistance calculated by the glucose infusion rate during a euglycemic hyperinsulinemic glucose clamp procedure is significantly correlated with the insulin-mediated net decrease in erythrocyte Na+ content (r = 0.58, P < 0.05), as well as with net increase in erythrocyte K+ (r = 0.64, P < 0.05) and Mg2+ (r = 0.67, P < 0.01) content and to basal diastolic blood pressure (r = -0.63, P < 0.05). We conclude that in elderly subjects the age-related and normally occurring insulin resistance might contribute to the increase of arterial blood pressure through its effect on cell cation content.