Effects of triiodothyronine on the insulin-like growth factor system in primary human osteoblastic cells in vitro.

Thyroid hormone plays a major role in the regulation of bone metabolism but the mechanism by which this is accomplished is not clear. Interactions of thyroid hormone with the growth hormone/insulin-like growth factors (IGFs) axis suggest an alternate pathway of action for triiodothyronine (T(3)) on bone formation, besides direct effects. The present study investigates the influence of T(3) on IGF-1, IGF-2, IGF-1 receptor (IGF-1R), and IGF binding protein (IGFBP) transcripts, and on IGF-1 action in human osteoblastic cells (hOB) under serum-free culture conditions. No influence of T(3) on IGF-1, IGF-2, IGFBP-3, or IGFBP-4 mRNA levels in hOB was observed. However, T(3) at concentrations of 10(-8) mol/L and 10(-7) mol/L increased IGF-1R mRNA levels in a dose-dependent manner (p < 0.01) and enhanced IGFBP-5 mRNA levels at a concentration of 10(-7) mol/L (p < 0.05), as assessed by reverse transcriptase-polymerase chain reaction. Correspondingly, Scatchard analysis of [(125)I]-IGF-1 binding revealed that T(3) at 10(-7) mol/L increased the number of IGF-1 binding sites in hOB, with small changes in receptor affinity. In addition, a synergistic effect of T(3) and IGF-1 on hOB proliferation was found (p < 0.05). We conclude that IGF-1R and IGFBP-5 are thyroid hormone target genes in human osteoblasts, whereas IGF-1 mRNA expression itself appears not to be regulated by T(3) in hOB. However, T(3) stimulates IGF-1R mRNA expression as well as IGF-1 binding and IGF-1 induced cell proliferation in osteoblasts, thus suggesting thyroid hormone may potentiate the effect of IGF-1 at the receptor level. This may contribute to the positive effects of thyroid hormone on bone formation, which, in addition, may be modulated by increased IGFBP-5 expression.

The effects of triiodothyronine on human osteoblast-like cells metabolism and interactions with growth hormone.

The expression of thyroid hormones receptors in osteoblasts and osteoclasts has involved these cells as direct targets for triiodothyronine (T3), but thyroid hormones may also interact with other hormones or local growth factors to exert their actions on bone cells. Among these, growth hormone (GH) is recognised as participating in the acquisition and maintenance of bone mass and exerting stimulatory effects on human osteoblastic cells. The aim of this study was to investigate T3 effects on primary human adult osteoblast-like cells (HOB) as well as to test for possible interactions between T3 and GH on bone cell metabolism. Primary human bone cell cultures were obtained by outgrowth from trabecular bone fragments from the hip and knee. Dose-response studies demonstrated enhanced [3H]-thymidine incorporation for T3 at 10(-9), 10(-8), 10(-7) and 20(-7) M, with a maximal response of 162.81 +/- 12.97 % with T3 10(-8) M, compared to vehicle (p < 0.001). Time-course studies showed an increased osteoblast-like cell proliferation after 24 h, followed by a decrease of cell proliferation by 48 h and 72 h of culture, respectively, when compared to control cells, with a maximal response after 72 h (T3 10(-10) M: 45.21 +/- 6.97 %, p < 0.01). In addition, T3 markedly increased specific alkaline phosphatase (AP) activity in HOB (10(-10) M: 169.86 +/- 12.14 % vs. control, p < 0.001), but no significant influence on type I procollagen propeptide (PICP) production was observed. At 10(-9) - 10(-7) g/ml, GH significantly enhanced HOB proliferation (p < 0.001) however, GH effects were not dose-dependent. Triiodothyronine, at a high concentration (10(-7) M), stimulated GH-receptor (GHR) mRNA levels by 165.20 +/- 16.54 % after 24 h (p < 0.05). Correspondingly, a synergistic effect of T 3 with the same concentration and GH on cell proliferation in human adult osteoblast-like cells was found.

Effects of parenteral pharmacological magnesium loading on insulin secretion in experimental thyrotoxicosis.

Thyrotoxicosis is characterised by decreased magnesium pool and also insulin resistance. The present study is evaluating the parameters of glucose metabolism under pharmacological magnesium loading in experimentally induced thyrotoxicosis, in rats. Insulin secretion was significantly increased in thyrotoxicosis compared to controls, expressing probably the status of insulin resistance due to thyroxine excess. After intraperitoneal magnesium infusion, plasma magnesium reached pharmacologically high concentrations and insulin secretion decreased significantly, but this decrease was not accompanied by alterations of glucose homeostasis. In controls, we also found a tendency towards the decrease of insulin secretion after magnesium loading, but it did not reach statistical significance. Thus, insulin secretion seems more sensitive to the inhibitory effects of magnesium overload in experimental thyrotoxicosis.

45Ca uptake in tooth germs: effects of parathormone, calcitonin and propranolol.

Tooth germs harvested from 6-day-old rats were incubated in a 45Ca-containing medium for 6 hours. The addition of PTH consistently stimulated the increase in 45Ca uptake, while calcitonin significantly reduced both the PTH-stimulated incorporation and the spontaneous one. Dibutyrul CAMP (3.5 mM) had no effects. Propranolol significantly inhibited the spontaneous and stimulated 45Ca uptake. Results obtained support the concept that the two hormones have a clear influence on calcium entering the tooth germs in full calcification process probably by early membrane effect.

Effect of thyroid hormones on osteolysis "in vitro".

The effect of triiodothyronine and thyroxine no the stimulation of the release of 45Ca was followed up in rat calvaria incubated "in vitro" and compared with the effect produced by parathormone. Both triiodothyronine and thyroxine stimulated osteolysis. The response was dependent on dose. Triiodothyronine may intensify the effect of parathormone if added to the incubation medium, whereas thyroxine does not exert the same effect. The results obtained demonstrated a well defined osteolytic effect of both thyroid hormones.

Effect of "APUD"-type hormones on osteolysis in vitro.

Besides their well-known actions, glucagon, ACTH, pentagastrin and insulin from the APUD series exert a direct action on the bone calcium content. Incubation with these substances of rat calvaria in vitro yields an evident stimulation of osteolysis with ACTH. Pentagastrin inhibits osteolysis. Glucagon and insulin inhibit parathormone-stimulated osteolysis, with no influence on the spontaneous one. Glucagon, resembling calcitonin, stimulates the 45Ca uptake from the incubation medium. The action of these substances completes the series of hormones influencing bone calcium metabolism, underlining possible interference actions of APUD-type hormones.

Reactivity to calcitonin after ovariectomy.

The hypocalcemic response to calcitonin injected in female rats disappeared after their castration. The substitution treatment with estrogens does no allow reestablishment of the hypocalcemic effect obtained in intact animals, a fact that lays stress on the role of the whole ovarian secretion in maintaining a normal response to calcitonin. The response of castrated animals to calcitonin calls attention to the conditional role of estrogens in validating the osteoprotecting effect of calcitonin.

Thyroparathyroid conditioning of hydrocortisone and glucagon-induced hypocalcemic effect in rats.

A single dose of 2.5 mg hydrocortisone induced hypocalcemia in both intact and parathyroidectomized rats. This effect disappeared in the thyroparathyroidectomized group and did not reappear after the substitution with parathormone. A single dose of 0.2 mg glucagon produced a significant hypocalcemic effect in intact, but not in parathyroidectomized rats. In the latter group, however, the hypocalcemic effect of glucagon reappeared after the administration of parathormone. It was concluded that the hypocalcemic effect of hydrocortisone may be conditioned by the presence of calcitonin secreting thyroid and that of glucagon - by the presence of parathyroids.

Effect of magnesium ion upon the calcitonin content of rat thyroid.

Thyroid calcitonin level was assayed in male rats after administration of magnesium gluconolactate. The resulting acute hypermagnesemia caused a significant decrease in calcitonin. However, calcemia showed unimportant changes, which suggests that the inhibitory action of magnesium is exerted directly on calcitonin secretion and is not affected by the calcium ion.

Insulin secretion in magnesium silicate-induced osteopenia in rats.

Local inflammation induced in rats by subcutaneous injection of magnesium silicate is followed by general metabolic, endocrine and immune reactions. Three weeks after inflammation induction, important bone loss, inflammation-mediated osteopenia (IMO) were found. The study proposes to investigate if IMO influences endogenous insulin secretion and if insulin therapy modifies the amount of bone loss, knowing its osteoprotective effect. Experiments were carried out on adult rats, three weeks after local inflammation induction. Baseline glucose and insulin levels did not change as compared to controls. After glucose stimulation, insulinogenic index decreased in the IMO group, which evidences decrease of insulin secretion. Insulin treatment initiated at the same time with IMO did not induce significant changes as regards the response to glucose tolerance test. However, bone calcium was increased as compared to the untreated IMO group, without reaching the control level.

Inhibitory role of somatostatin on calcitonin secretion.

Calcitonin (CT) secretion is not exclusively controlled by calcemia, but the secretory tonus is maintained by the beta-stimulatory adrenergic system Somatostatin (SMS) plays a neuromodulatory role with the reduction of CT secretion by its interference at the central and peripheral level of the beta adrenergic receptors. The experiments were carried out on groups of rats in which the effect of SMS on CT content of the thyroid gland was followed up. Thus, SMS administered i.c.v. significantly reduced the basal CT secretion without blocking the stimulatory effect of calcium. The results were comparable with those obtained after the blockade of the sympatho-adrenergic system by chemical sympathectomy with 6HODA or propranolol. Central blockade of alpha receptors with phentolamine determined a significant rise of CT. This effect was annihilated by SMS. The i.v. administration of SMS did not induce a change in CT content of the thyroid, but blocked the stimulatory action of hypercalcemia. The results are identical with those obtained by blocking the beta-receptors with propranolol. SMS also blocked the stimulatory effects of isoproterenol on CT secretion. The data obtained revealed the fact that SMS lowers CT secretion by the central and peripheral interference of the sympatho-adrenergic path, maintaining the secretory tonus of the thyroid C cells.

Effect of calcitonin on phosphorylase a and glucose-6-phosphatase activities in the hepatic particulate glycogen of rats.

A single dose of calcitonin (CT) (70 mU MRC/100 g body weight) was administered in 50 and 90 days old male Wistar rats. In both ages CT determined a rise of Phosphorylase a and glucose-6-phosphatase activity as well as an increased calcium accumulation at the level of liver particulate glycogen. In the rats receiving CT glycemia increased and the liver glycogen content decreased. With the exception of liver glycogen the other metabolic parameters studied were modified at the two ages in the same manner under the influence of the same dose of CT.

Calcitonin reserve and the differentiated response to calcitonin in osteoporosis.

The authors followed up the effect of calcium and calcitonin administration on phosphorus-calcium balance and on calcium retention, in correlation with the level of endogenous calcitonin, in 20 patients of perimenopause age (40-50 years), with radiologically confirmed osteoporosis. Calcium retention after loading with calcium gluconate (180 mg in i.v. injections) was determined before and after administration of salmon calcitonin (100 IV in i.m. injections). Two types of responses were noted. In a group of patients calcitonin administration determined a rise of calcium retention at the same time with the improvement of other biochemical parameters as well as a normal calcitonin response. The presence of high levels of circulating calcitonin 24h after loading with exogenous calcitonin demonstrated a slower inactivation rate of the hormone in these patients. The other group showed no positive response to calcitonin. The proposed test is a criterion for the selection of the patients with osteoporosis in view of chronic calcitonin treatment.

Stimulating effect of calcium channel activator BAY-K 8644 on calcitonin secretion.

The calcium channel activator BAY-K 8644 injected intravenously produces a significant rise in the calcitonin content of the thyroid. Because adrenalin and noradrenaline play a certain role in the regulation of ionic calcium channels and in the preservation of the secretory tonus of the calcitonin secreting C cells, the effect of BAY-K 8644 was followed-up in adrenalectomized animals and in animals in which propranolol was previously administered. Adrenalectomy of beta receptor blocking does not prevent the effect of BAY-K 8644 on the activation of calcium channels, the calcitonin secretion being stimulated in these conditions too. The data obtained stress the relative independence of the calcium channels or the beta-adrenergic stimulating system in the achievement of calcium ion transfer. However, the calcitonin levels obtained were lower in adrenalectomized animals or following propranolol after stimulation with BAY-K 8644 in those with intact sympatho-adrenergic tonus. This stresses the importance of the integrity of the beta receptors in the activation of the calcium channels.

Changes in calcitonin secretion and in serum magnesium following adrenalectomy in rats.

The level of serum magnesium and also thyroid of calcitonin content was followed up in the conditions of adrenalectomy after hydrocortisone replacement as well as after calcium propranolol administration. Six days after adrenalectomy a significant decrease both of calcitonin secretion and of serum magnesium was noted. Hydrocortisone replacement reestablished the levels of both parameters to the levels found in controls. Adrenalectomy also reduced the calcitonin secretory response consequent to calcium loading, but calcium excess simultaneously determined hypermagnesemia. Propranolol, a beta blocker with direct action on the secretory tonus of the thyroid C cells, completely annulled the stimulation effect of calcium in adrenalectomized animals but not the hypermagnesemic response. The adrenocortical hormones interfere in the calcitonin-magnesium secretory antagonism, together with the sympathetic -adrenergic system, ensuring the secretory control of calcitonin especially by blocking the occurrence of an excess of magnesium which reduces calcitonin secretion.

Adrenal function in early and metastatic breast cancer: dexamethasone suppression of plasma cortisol.

Plasma cortisol, 17-hydroxyprogesterone (17-OH-P), progesterone, FSH, LH and prolactin were determined by RIA, in 14 cancer patients without metastases aged between 40 and 74 years (6 cases of breast cancer: T123, N01, M0 and 8 with other forms of cancer). The cancer patients were investigated: (A) under basal conditions, (B) after three days' adrenal suppression by dexamethasone, 3 mg/day and (C) immediately after local radiation therapy (4500 rads). The basal mean hormonal values in these patients showed increased cortisol, decrease 17-OH-P and normal values of progesterone, FSH, LH and prolactin. Plasma cortisol was significantly reduced by adrenal suppression with a percentage reduction of 19.44 without differences between breast cancer patients and patients with other forms of cancer; adrenal suppression induced an increase of 17-OH-P only in male cancer patients. The only significant hormonal changes after local radiation therapy were increased plasma 17-OH-P values in both female and male cancer patients. A second group investigated in the present study consisted of 11 patients (6 castrated and 5 postmenopausal women) with metastatic breast cancer and presented increased plasma cortisol values, decreased 17-OH-P values, and a great scatter in the estrone values, some of them being very high.

Calcitonin secretion in hyperthyroidism.

The level of circulatory calcitonin was determined by the RIA method in 45 hyperthyroid patients in evolution, before and after calcium stimulation or after pentagastrin injection. The basal calcitonin secretion was increased in only 7 of the 45 patients studied, exceeding 500 pg/ml. After a slow calcium loading the mean of calcitonin increased, but with variable individual values. The rapid calcium loading was not followed by significant increases after 30 minutes. After pentagastrin a secretory peak of over 1,800 pg/ml was obtained in 4/5 cases. Results point out the state of the calcitonin-secreting system in hyperthyroidism and its possible role in the skeleton protection in these patients.

The effect of some enkephalins on calcitonin secretion.

Synthetic enkephalins: 5 metenkephalin, D2 proenkephalin and 5 leuenkephalin and morphine were injected into cerebral ventricles of rats. After 30 minutes the rats were sacrificed and the calcitonin content of the thyroid was assayed. As compared to the controls, morphine reduced significantly the secretion of calcitonin. Of the enkephalins, only 5 leuenkephalin had a borderline effect on calcitonin secretion, stimulating to a significant degree the rise of the hormone. The data obtained demonstrate the selective effect of opiates on calcitonin secretion.

Calcitonin secretion in rats with inflammation mediated osteopenia.

The authors followed up the occurrence of inflammation-mediated osteopenia (IMO) in young and adult rats weighing 50 g and 150 g, respectively. The calcemia, the amount of bone calcium and the calcitonin content of the thyroid gland were determined. Calcium loss from bones was similar in both groups, irrespective of age. However, in the young animals a significant rise of calcitonin content of the thyroid gland was found (11.08 ng/ml as compared with 3.81 ng/ml in the controls). In adult animals with IMO the level of calcitonin in the thyroid gland showed no change as compared with the controls. Calcitonin reactive hypersecretion in the young animals was not able to prevent the development of IMO, characterized by an important loss of bone calcium.