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Serum BNP (brain naturiuretic peptide) and ANP (atrial natriuretic peptide) levels are reportedly elevated in patients with thyrotoxicosis. The increases may not be due to thyrotoxicosis itself but to secondary cardiovascular changes such as chronic heart failure (HF) or atrial fibrillation (AF) which frequently accompany thyrotoxicosis. We measured serum ANP and BNP levels in 130 patients with thyrotoxicosis and correlated them with HF severity and thyroid function. Thirty-seven normal subjects served as controls. Serum BNP levels in thyrotoxic patients were significantly higher than those in control subjects and significantly correlated with serum free T4, free T3 and ANP levels. In untreated Graves' disease serum BNP level was significantly elevated in patients with HF or AF. Multiple regression analysis revealed that HF, free T4, female gender and AF are independent contributing factors to the elevated BNP level, and that these four factors contributed about 40%. On the other hand, HF and AF were contributing variables for ANP level but the overall contribution of these factors was only 10%. After normalization of thyroid function, serum BNP levels were normalized in 70.5% of Graves' patients. BNP level in euthyroid state was dependent on the presence of HF and the BNP value before therapy, but not on thyroid hormone levels or AF. These data suggest that the cardiovascular condition is the major factor responsible for the elevated serum BNP and ANP levels in thyrotoxic patients, while thyrotoxicosis itself is an independent but minor contributing factor. Thus, the determination of serum BNP levels in thyrotoxic patients is useful for monitoring cardiovascular conditions of HF.

It has been reported that adult patients with growth hormone deficiency (GHD) have increased risk factors for cardiovascular disorders in Western countries. However, data on this issue in Japan have not been fully investigated. To clarify the situation on this issue in Japan, a nationwide questionnaire survey was conducted by Study Group of Hypothalamo-Pituitary Disease, and medical records of 863 adult patients were recruited. The incidences of complications and risk factors for cardiovascular diseases were compared between GH deficient patients (GHD, n = 494) and GH intact patients (GHI, n = 369). The incidence of myocardial infarction was higher in GHD (1.2%) than in GHI (0.8%), but not significantly. The incidences of angina pectoris and liver dysfunction were significantly higher in GHD than in GHI (2.8 vs 0.8%, p = 0.048, and 11.9 vs 5.5%, p<0.0001, respectively). The prevalences of obesity and hyperlipidemia (HL) were significantly higher in GHD (p = 0.004, p<0.001, respectively). Combinations of HL plus diabetes mellitus (DM) or hypertension (HT) were more common in GHD than in GHI (HL + DM; 7.2 vs. 2.9%, HL + HT; 10.9 vs. 2.9%). These results indicated that GH deficiency have an increased prevalence of cardiovascular risk factors in Japanese similar to Western countries, and suggested that GH supplement therapy may be needed to reduce those complications.

Leptin receptors are distributed throughout the body and leptin has been shown to have various effects. As we have recently demonstrated a positive correlation between serum leptin levels and TSH in euthyroid subjects, we investigated the effect of leptin on the thyroids. It was observed that serum leptin levels were negatively correlated with free thyroxine/TSH ratios in the serum of euthyroid female subjects. This suggests that leptin may modulate TSH effects. RT-PCR for leptin receptor expression revealed that FRTL-5 cells possess the gene transcript to the long cytoplasmic form of the receptor. Leptin actually appeared to induce an increase in c-fos mRNA expression. However, it inhibited iodide uptake typically induced by both TSH and dibutyryl cAMP, while leptin did not inhibit TSH-induced cAMP production or TSH-stimulated DNA synthesis in 4H medium (in the absence of insulin and TSH). Leptin also was observed to inhibit TSH- and dibutyryl cAMP-induced Na+/I- symporter and thyroglobulin mRNA expression. Lastly, leptin was seen to inhibit TSH-stimulated thymidine incorporation in 5H medium. Taken together, these results suggest that leptin suppresses TSH-induced thyroid function. Therefore, we hypothesized that leptin may be one of the regulators of thyroid function in obese patients.

Although a number of abnormalities in oncogenes have been reported in thyroid neoplasms, little information is available on the signal transduction pathway involved in neoplastic thyroid cell growth. Both p70S6 kinase (p70S6K) and Akt are kinases downstream of phosphatidylinositol 3 kinase (PI3K). These kinases are phosphorylated and activated by growth factors including IGF-1, EGF/TGF-alpha, and HGF in thyroid cells. Since the receptors for these growth factors are reportedly overexpressed in human thyroid cancer, we hypothesized that the PI3K-mediated signalings are overactivated in thyroid cancers. Tumorous and adjacent normal tissues of 20 patients with papillary thyroid cancer were obtained at surgery, and expression of p70S6K and Akt were measured by Western blot. Expression of the protein levels of p70S6K was increased in tumor tissues (T) compared to normal thyroid tissues (N), and expression of phosphorylated p70S6K was also significantly increased in tumor than in surrounding normal tissues. Overexpression of p70S6K in tumor tissues was further confirmed by immunohistochemistry. Strong immunoreactivity in the cytoplasm of thyroid cancer cells was seen in the majority of cases, whereas little immunoreactivity was found in the surrounding normal portion. Expression of phosphorylated Akt (pAkt) was also significantly higher in tumor tissues. Phosphorylation of Bad (pBad), a substrate of Akt, was also increased in the tumor tissues in association with activation of Akt, and the T/N ratio for pAkt positively correlated to the T/N ratio for pBad. The data presented here demonstrate that both p70S6K and Akt are activated in the majority of human papillary cancer cells. Activation of these signalings may be involved in the progression of papillary carcinoma by stimulating cell proliferation and/or preventing apoptosis.

Transsphenoidal surgery is the treatment of choice for ACTH-producing pituitary adenoma (Cushing's disease) and pituitary irradiation is widely considered the most appropriate treatment for patients with Cushing's disease for whom transsphenoidal surgery has been unsuccessful. We studied 49 consecutive patients who underwent transsphenoidal surgery for the treatment of Cushing's disease at Tokyo Women's Medical University from 1977-1997 with a mean follow-up duration of 87.6 months (range, 24-253 months). We examined the relationship between postoperative endocrinological data, assessed between 3 and 8 weeks after surgery, and long-term outcome and efficacy of pituitary irradiation after surgery. Long-term remission was defined as the regression of the symptom and signs of Cushing's syndrome, and restoration of normal levels of plasma ACTH, cortisol and urinary free cortisol, together with adequate suppression of morning plasma cortisol levels following the administration of low dose (1 mg) of dexamethasone. Thirty patients had no additional treatment after pituitary surgery. Only 1 of 25 patients (4%) whose postoperative plasma cortisol level was less than 2 microg/dl developed recurrent disease whereas 3 out of 5 patients with postoperative plasma cortisol levels higher than 2 microg/dl relapsed. Postoperative external pituitary radiation was used to treat the remaining 19 patients. Four patients who received radiation therapy had a low or undetectable postoperative plasma cortisol level (<2 microg/dl, 56 nmol/L) and all of these patients developed hypopituitarism whereas 5 patients with subnormal plasma cortisol levels (2.0-10.0 microg/dl) remained in remission. Among 10 patients with persistent disease after surgery, 6 entered remission 6-47 months after irradiation but one of them subsequently relapsed after 108 months. These results suggest that 1) additional therapy should be avoided in patients with a postoperative plasma cortisol less than 2 microg/dl because relapse is very rare and radiotherapy will frequently induce hypopituitarism, 2) patients with a subnormal cortisol level following surgery should be treated with pituitary irradiation, because the relapse rate is reportedly high and radiotherapy is effective in preventing relapse, 3) radiotherapy in patients with persistent disease after surgery is effective only in 50% (5/10) of the patients.

The 20K hGH isoform is produced by alternative splicing of GH mRNA, and comprises approximately 10% of all GH in the pituitary. The physiological role of 20K hGH remains to be determined partly because of the lack of a simple and specific assay. We have established sensitive enzyme-linked immunoadsorbent assays (ELISAs) specific to 20K and 22K hGH. The serum levels of 20K hGH after overnight fasting was 118 +/- 178 pg/mL (N=282) in normal women, significantly higher than in normal men (64 +/- 170 pg/mL, N=226). However, there was no difference in the proportion of 20K hGH to 20K plus 22K hGH between men (6.3 +/- 2.6%, N=176) and women (6.3 +/- 2.1%, N = 263). No correlation was detected between the ratio of 20K hGH and age, body height, body weight or body fat mass in normal subjects. The proportion of 20K hGH was significantly (P < 0.001) higher in patients with active acromegaly (9.2 +/- 2.2%, N=33) and in patients with anorexia nervosa (9.0 +/- 1.9, N=8), both of which are characterized by chronic elevation of circulating GH levels. The proportion of the 20K hGH in successfully treated acromegalic patients did not differ from that in normal subjects, suggesting that GH-producing pituitary tumors secrete a higher proportion of 20K hGH, or chronic excess of 22K hGH altering the metabolic clearance rate of 20K hGH. The values in patients with adult growth hormone deficiency (GHD), hyperthyroidism, primary hypothyroidism, or GH-independent short stature did not differ from those in normal subjects. The 20K ratio did not change after acute GH provocative tests such as insulin tolerance test and GRH test. These results suggest that secretion of 20K hGH from the pituitary is under the same control as that of 22K hGH.

We investigated whether thyroid status modulates serum leptin concentrations and body composition as determined by bioelectric impedance analysis (BIA). The percent body fat mass (%FM) in male Graves' disease was significantly lower than that in age- and sex- matched normal subjects, at the levels of 11.4+/-6.4% (mean+/-SD) vs 19.9+/-9.2% for men (n=12, P<0.05) but not for women (22.6+/-7.6% vs 24.9+/-13.1%, n=28). In contrast, in female hypothyroidism (n=11) %FM was significantly higher than that in normal subjects (32.9+/-11.5%, P<0.01). Among other body composition parameters, the percentage of body water (%BW), and lean body mass (LBM) were significantly lower in hypothyroid patients, and the ECM (extracellular mass)/BCM (body cell mass) ratio was significantly (P<0.0001) increased in Graves' disease which was the result of marked depletion of BCM with concomitant expansion of ECM. The serum leptin levels were significantly decreased in male Graves' patients (2.3+/-0.7 ng/ml, P<0.05), whereas in female Graves' patients (8.8+/-5.9 ng/ml) and patients with hypothyroidism (9.5+/-7.6 ng/ml), the levels were not different from those of normal controls matched for BMI or %FM. There was a positive correlation between serum leptin levels and %FM in female Graves' patients (r=0.635, P=0.001) and in hypothyroid patients (r=0.801, P=0.014) but not in male Graves patients. There was no significant relationship between serum leptin levels and thyroid hormones, TRAb, or TSAb. In euthyroid obese subjects there was a positive relationship between serum leptin levels and serum TSH levels (r=0.37, P<0.01). These results suggest that hyperthyroidism is characterized by the decreased fat mass and serum leptin levels in men, but female patients appear to be resistant to the effect of thyroid hormones. Together with previous reports, thyroid status has a minor role in the regulation of serum leptin levels.

Bioactivity of Insulin-like growth factors (IGEs) are positively or negatively regulated by IGF binding proteins (IGFBPs). Like IGFs, production of IGFBPs are influenced by a number of hormonal factors. We studied the effects of cytokines on production of IGFBPs in human fibroblasts in culture. Both IL-1beta and TNF-alpha inhibited IGFBP-3 (42/38 KDa species) production in a concentration dependent manner judged by Western ligand blot. Expression of IGFBP-3 mRNA was also decreased by these cytokines. Moreover, the treatment with IL-1beta and TNF-alpha resulted in appearance of smaller mol weight (26 KDa) immunoreactive IGFBP-3 fragment(s) which lacked the ability to bind 125I-IGFs, indicating that these cytokines degrade IGFBP-3 via activation of proteases. Both IL-1beta and TNF-alpha decreased production of IGFBP-4, whereas they increased that of IGFBP-6, IL-6 had little effect on production of IGFBPs. Likewise, interferon-gamma failed to affect of production of IGFBPs except at high concentrations. The present data demonstrate that cytokines, especially IL-1beta and TNF-alpha are potent regulators of IGFBPs production and degradation. These cytokines may alter tissue uptake of IGFs and help to counteract the catabolic state induced by them.

In order to identify the mutual interaction between GH and leptin, we studied the effect of GH on fatty Zucker rats. GH administration at a high dose (5.0 IU/kg) reduced % body fat after 7 days. The leptin mRNA level in subcutaneous fat tissue was not changed but that in epididymal fat tissue was decreased by an even lower dose of GH (1.5 IU/kg). IGF-I treatment (200 microg/kg/day) did not change the % body fat or leptin mRNA level. These observations suggest that GH directly interacts with visceral fat and reduces fat mass and leptin expression. We also measured serum leptin levels in patients. The levels in patients with acromegaly were significantly lower than those in normal subjects with the same amount of body fat, but serum IGF-I and urinary C peptide excretion rates were higher in the acromegalic. These observations also suggests that GH directly interacts with adipose tissue and reduces leptin expression. Next we investigated the direct action of leptin on GH release from the pituitary. Leptin pretreatment of pituitary cells in culture or rats in a fasted or fed condition did not change GRH induced GH secretion. As indicated also by other recent studies, leptin may increase GRH or decrease somatostatin secretion by the hypothalamus. Thus GH interacts with fat tissues and leptin may be a good marker of the interaction.

The Foundation for Growth Science has been controlling the use of GH by its registration system, which includes a scoring system for the eligibility for GH treatment according to the diagnostic criteria for GH deficiency (GHD) established by the Study Group for Hypothalamo-pituitary Disorder of the Ministry of Health and Welfare. Until 1995, 28,876 patients with GHD (19,432 boys and 9,444 girls) had been registered as eligible for GH treatment. The number of patients registered in a year increased gradually till 1990 due to the unlimited hGH supply by recombinant techniques and the change in the criteria for GH treatment and the number registered became stable after 1990. The frequency of GH-treated patients is calculated to be 55.2/100,000 persons (72.2/100,000 in boys and 37.1/100,000 in girls) in patients born between 1960 and 1990. The highest frequency was 148.4/100,000 persons (191.7/ 100,000 boys and 103.7/100,000 girls) in 1981, when 2,278 patients (1,508 boys and 770 girls) were born. Eligibility for GH treatment is assessed according to the scoring system which is basically dependent on peak GH values in provocation tests so that standardization of GH values measured with the various commercial GH kits is required to avoid inequality of patients' access to the treatment. In samples obtained by GRF test in 10 normal volunteers, hGH was measured with seven human GH (hGH) kits at a laboratory center. Since the RIA value has been used historically for the diagnosis of GHD, the mean of two RIA measurements was selected as the basis for the standardization procedure and the linear regression formula was used for each hGH kit. After the freely available supply of hGH obtained by recombinant DNA techniques, the role of the Foundation for Growth Science has changed to avoid hGH abuse. Even with this regulation, the frequency of registered patients may indicate a tendency to GH overuse.

GH binding to its receptor, which belongs to the cytokine receptor superfamily, activates Janus kinase (JAK) 2 tyrosine kinase, thereby activating a number of intracellular key proteins such as STAT (signal transducers and activators of transcription) proteins and mitogen-activated protein (MAP) kinases, which finally lead to GH's biological actions including gene expression. In contrast to receptor tyrosine kinases, the signalling pathways leading to MAP kinase activation by GH are poorly understood but appear to involve Grb2 and Shc. We now show that GH stimulated tyrosine phosphorylation of epidermal growth factor receptor (EGFR) and its association with Grb2, and concomitantly stimulated MAP kinase activity in liver, a major target tissue. Expression of EGFR and its mutants into CHO-GH receptor (GHR) cells revealed that GH-induced full activation of MAP kinase and c-fos expression required tyrosine phosphorylation sites of EGFR but not its intrinsic tyrosine kinase activity. Moreover, by also using dominant negative JAK2 and in vitro kinase assay, we demonstrated that tyrosine 1068 of EGFR was evidently one of the major phosphorylation and Grb2 binding sites stimulated by GH via JAK2. These data suggest that the role of EGFR in GH signalling is to be phosphorylated by JAK2, thereby providing docking sites for Grb2 and activating MAP kinases and gene expression. This novel cross talk pathway may provide the first example of the hormone and cytokine receptor superfamily transducing signals via associated nonreceptor tyrosine kinase by phosphorylating growth factor receptor and utilizing it as a docking protein independent of its receptor tyrosine kinase activity.

We measured serum levels of the carboxy-terminal propeptide of type 1 procollagen (P1CP) as a marker of bone formation and the carboxy-terminal telopeptide of type 1 collagen (1CTP) as a marker of bone resorption by RIA in sera from 40 Graves' disease patients and 14 Hashimoto's disease patients before and during treatment. The serum P1CP levels of the untreated Graves' disease were significantly higher than in the controls (176.8 +/- 93.5 vs. 107 +/- 35 ng/ml, P < 0.01), and these levels decreased significantly during treatment with antithyroid drugs. There was a significant statistical correlation between serum P1CP levels and serum total alkaline-phosphatase activity (r = 0.61, P < 0.01) in the patients with Graves' disease and Hashimoto's disease as a whole. 1CTP levels were also significantly increased in untreated Graves' patients (6.5 +/- 2.8 compared with 2.7 +/- 1.1 ng/ml in normal subjects, P < 0.01). The P1CP/1CTP ratio, which reflects the relative ratio of bone formation to bone resorption, was lower than normal in untreated Graves' disease, but increased following the normalization of thyroid function. The results of this study suggest that the measurement of serum P1CP and 1CTP levels may be useful in evaluating bone metabolism in thyroid disease.

To evaluate the role of insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) in excessive fetal growth (macrosomia) in diabetic pregnancy, 84 insulin-treated diabetic mothers and their infants were tested for serum concentrations of IGF-I, IFG-II, and IGFBP-1, -2 and -3. These parameters were correlated with the birth weight of neonates and placental weight. IGF-I and II levels were determined by specific radioimmunoassays (RIAs) after serum samples were extracted with aid-ethanol. IGFBPs were measured by Western immunoblot with specific antibodies to the respective IGFBP species. Serum concentrations of both IGF-I and IGF-II in mothers with either IDDM or NIDDM increased with the gestational period, reached a plateau at the third trimester, and returned to non-pregnant levels within 7 days after delivery. These values were not different from those in normal mothers before and throughout pregnancy. As previously reported, IGF-I concentrations in cord serum of neonates born to diabetic mothers were (P < 0.01) higher than those of newborns of normal mothers. Likewise, cord blood IGF-II levels were 2-fold higher in babies of diabetic mothers (P <0.001). Fetal IGF-I and IGF-II correlated with each other and with maternal HbA1C, and they positively correlated with either birth weight or placental weight. Cord IGFBP-3 concentrations were significantly higher in diabetic pregnancy, but IGFBP-2 concentrations were not different from those in normal pregnancy. Cord IGFBP-1 concentrations were significantly higher only in babies of mothers with IDDM. None of these cord IGFBP concentrations correlated with birth weight or placental weight. The data suggest that fetal IGF-II, like IGF-I, is involved in fetal and placental growth in diabetic pregnancy. The role of IGFBPs remained to be determined.

A 76-year-old female patient who had been taking vitamin D2 100,000 U/day for more than 14 years due to hypoparathyroidism following total throidectomy was admitted because of protracted hypercalcemia. On admission, the levels of serum vitamin D2 (99.8 ng/ml) and 25-OHD2 (356 ng/ml) were very high, and 1,25-(OH)2D2 was low (4.0-18.7 pg/ml). Serum D3' 25-OHD3 and 1,25-(OH)2D3 were below the normal range. Despite intensive hydration with saline, intravenous hyperalimentation with phosphate- and calcium-free nutrients, and administration of glucocorticoid and calcitonin, the hypercalcemia persisted, accompanied by hypoproteinemia, edema, pleural effusion and congestive heart failure. The serum D2 and 25-OHD2 concentrations remained high and were accompanied by a gradual increase in 1,25-(OH)2D2 (121 pg/ml), which further increased after the administration of bisphosphonate (pamidronate) to 183 pg/ml. Seventeen months later, serum calcium and 1,25-(OH)2D2 were normalized but serum D2 and 25-OHD2 remained high. The serum 24,25-(OH)2D2/25-OHD2 ratio was relatively constant throughout her clinical course, whereas the low serum 1,25-(OH)2D2/25-OHD2 ratio at admission gradually increased during admission, suggesting that the increase in serum 1,25-(OH)2D2 is due to increased production rather than decreased degradation. The administration of pamidronate further increased serum 1,25-(OH)2D2. These features of the clinical course demonstrate that the 1,25-dihydroxyvitamin D concentration in hypercalcemic patients with protracted vitamin D intoxication may be decreased, normal or increased. Possible factors responsible for a protracted increase in serum 1,25-(OH)2D2 are body weight loss, hypoproteinemia, and phosphate depletion. In addition, some bisphosphonates would certainly promote PTH-independent production of 1,25-(OH)2D2.

A 65-year-old female patient was admitted with complaining chiefly of lower back pains and arthralgia in the bilateral knee joints of 10-years duration. The serum calcium concentration was normal or only slightly increased, whereas the serum intact PTH and 1,25-dihydroxyvitamin D concentrations were substantially increased. Serum phosphate and 25-hydroxyvitamin D concentrations were decreased. Renal function was normal. Serum alkaline phosphatase activity, the osteocalcin concentration and urinary hydroxyproline excretion were markedly increased. Bone X-ray examination showed severe osteopenia and bone biopsy revealed hyperosteoidosis without tetracycline deposition, consistent with osteomalacia. A parathyroid adenoma was demonstrated by echography and CT-scan. Surgical exploration of the neck revealed a chief cell adenoma behind the right upper pole of the thyroid gland. After parathyroidectomy, all the abnormal biochemical data gradually normalized and the patient has been doing well without any symptoms for the last 13 months. These clinical data suggest that osteomalacia of the patient was probably induced by hypophosphatemia of prolonged duration. When hypercalcemia is not evident in a patient with primary hyperparathyroidism, in whom serum alkaline phosphatase and intact PTH levels are inappropriately increased, osteomalacia should be taken into consideration.