In FRTL-5 cells, we and others have shown that TSH and insulin-like growth factor-I (IGF-I) stimulate DNA synthesis synergistically. The present study was undertaken to determine whether interaction between TSH and IGF-I also affects protein synthesis in this cell line, and if so by what mechanism. Quiescent cells were treated with TSH and/or IGF-I and [^3MH]valine incorporation into the acid-insoluble fraction was measured as a parameter of protein synthesis. Similar to their effects on cell proliferation, TSH or IGF-I alone induced protein synthesis only slightly, but treatment with a combination of TSH and IGF-I (or insulin with about a 100-fold higher concentration than IGF-I) greatly increased protein synthesis. The presence of IGF-I potentiated a TSH-concentration-dependent increase in protein synthesis and in DNA synthesis. In addition, we observed this potentiation when the cells were treated with other cAMP-generating agents and cAMP analogues instead of TSH. We have shown that priming with TSH potentiated DNA synthesis induced by IGF-I, whereas pretreatment with IGF-I enhanced protein synthesis induced by TSH. This observation suggested that protein synthesis and DNA synthesis were potentiated through different mechanisms. From an analysis of CAMP production, it appears that the potentiation of protein synthesis may be explained by an IGF-I-dependent increase in cAMP production induced by TSH at least in part. On the other hand, IGF-I and TSH stimulated (α-aminoisobutyric acid (AIB)) uptake synergistically, but RNA synthesis induced by IGF-I was depressed by TSH. From these results, we concluded that in FRTL-5 cells, IGF-I potentiated protein synthesis induced by TSH by means of complex mechanisms and the interaction between IGF-I and cAMP-dependent pathways may also have a physiological meaning in regulating protein anabolism.

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It has been proposed that high plasma free fatty acid (FFA) levels observed in patients with non-insulin dependent diabetes mellitus (NIDDM) contribute to the development of their insulin resistance. We examined patients with NIDDM to find whether maintaining plasma FFA levels in the fasting range with a euglycemic hyperinsulinemic clamp combined with an oral glucose load (clamp OGL) would affect insulin-mediated peripheral glucose uptake (PGU) and splanchnic glucose uptake (SGU). Nine NIDDM subjects (age, 55±3 years; duration of diabetes, 11±2 years; body mass index, 21.0 ±0.4kg/m²; hemoglobin A₁c, 9.0±0.3%; fasting plasma glucose, 9.4±3.0mmol/l, means±SEM) were hospitalized and treated with diet, oral hypoglycemic agents or insulin for at least 2 weeks to maintain fasting plasma glucose <8mmol/l. All the patients were subjected to two different protocols in a random order. On one protocol, under the hyperinsulinemic condition, FFAs were maintained at the their fasting levels (1.19±0.08) by triglyceride emulsion infusion (Lipid infusion study, L), and on the other protocol, FFAs were made to fall (0.26±0.06mmol/l) with saline instead of triglyceride emulsion infusion (Saline infusion study, S). During euglycemic (L, 5.4±0.2; S, 5.1±0.2mmol/l) hyperinsulinemic (L, 1377±108; S, 1328±67pmol/l) clamp, high FFA levels significantly reduced PGU (L, 26.7±3.6; S, 32.1±3.4μmokg^-1•min^-1, P<0.05) and SGU (L, 12.1±4.2; S, 27.5±5.6%, P<0.05). In conclusion, high FFA levels in patients with NIDDM impaired insulin-mediated glucose uptake in the splanchnic as well as peripheral tissues.

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Gonadotropin-releasing hormone (GnRH) and its agonists have been known to directly affect steroid hormone production in human granulosa cells. In this study, we examined effects of GnRH on Ca²⁺ mobilization, protein kinase C activity and steroidogenesis of human granulosa cells. Human granulosa cells were harvested by aspiration of follicles during oocyte retrieval for IVF. Test substances, that is, GnRH agonist ([D-Ser(TBu) ⁶]-LHRH-EA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), A23187, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and nordihydroguaiaretic acid (NDGA) were added to HTF medium with human granulosa cells. At the end of a 5-h incubation period, progesterone and estradiol in the medium were extracted and measured. GnRH agonist (10^-9M and 10^-8M) significantly stimulated (P<0.05) progesterone and estradiol accumulation in the medium. TPA (10^-7M, 10^-8M and 10^-9M) also significantly stimulated (P<0.05) progesterone accumulation. A23187 (10^-6M) significantly stimulated progesterone accumulation (P<0.05) and significantly enhanced (P<0.05) the stimulatory effect of TPA on progesterone production. H-7 (10^-3M, 10^-4M and 10^-5M) and NDGA (10^-3M, 10^-4M and 10^-5M) significantly inhibited (P<0.05) the effect of GnRH agonist on progesterone and estradiol production by the granulosa cells. The granulosa cells were loaded with 4μM Fura 2-AM, and the ratio of the intensities of fluorescent emission at 510nm with excitation at 340 and 380nm was calculated at 100-ms intervals. GnRH agonist (10^-7M and 10^-6M) increased (P<0.05) [Ca²⁺]i significantly as compared with the control. These results suggested that the effects of GnRH on progesterone and estradiol production in human granulosa cells were mediated partially by calcium mobilization and partially by C-kinase activity, and protein kinase C, [Ca²⁺]i and lipoxygenase were suspected of being important intracellular messages for GnRH in human granulosa cells.

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To determine the relationship between the serum levels of Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and those of bone formation and resorption markers in prostate cancer patients, we measured the serum levels of the cytokines and examined their relationship to biochemical markers of bone turnover in 46 untreated patients with prostate cancer. The carboxy-terminal propeptide of type I procollagen (PICP) levels were used as a parameter of bone formation, and the carboxy-terminal telopeptide of type I collagen (ICTP) levels were used as a marker of bone resorption. The relationship of these markers to the degree of bone metastasis was also examined. The serum levels of IL-6, PICP, ICTP, and prostate-specific antigen (PSA) were significantly higher in the patients with prostate cancer with bone metastasis (n=23) than in the patients without bone metastasis (n=23). The serum levels of TNF-α in approximately 85% of the patients were under the detectable limit (5pg/ml). The serum levels of IL-6 were not correlated with those of PICP or ICTP, but were related to the extent of bone metastasis. These results indicate that among patients with prostate cancer, IL-6 and TNF-α may not play major roles in the increased bone resorption in the patients with metastatic spread to bone. Our study thus demonstrated that the serum levels of IL-6 are closely related to the metastatic burden to osseous tissue in prostate cancer patients.

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Alendronate (4-amino-1-hydroxybutylidene-1, 1-bisphosphonate) is a potent inhibitor of bone resorption. The efficacy and safety of 36 weeks of treatment with alendronate were evaluated in Japanese women with osteoporosis, osteoporotic osteopenia or artificial menopause. The bone mineral density (BMD) of the lumbar vertebrae, markers of bone and calcium metabolism and clinical symptoms were monitored. A total of 113 randomly selected patients with osteoporosis or osteopenia were enrolled in the study, of whom 12 were excluded from the analyses because of lack of data. As a result, 101 patients were evaluated for the safety of the drug. Since eight patients were excluded from the efficacy analysis, 93 were evaluated. The incidence of adverse effects in the placebo (P), alendronate 2.5 mg/day (L) and alendronate 10mg/day (H) groups increased with increasing dose of alendronate, being 6.1, 14.3 and 18.2%, respectively. The most common adverse effects were gastrointestinal symptoms, none of which was serious. Lumbar BMD increased after 36 weeks of drug administration to 5.21%, 5.64% and -0.90% in the L, H and P groups, respectively (P<0.001, L vs. P and H vs. P). Serum alkaline phosphatase activity, serum osteocalcin and urinary deoxypyridinoline excretion were significantly decreased in a dose-related manner. Serum calcium and phosphorus were also significantly decreased after alendronate administration. Serum intact PTH was transiently increased. The present results indicate that alendronate effectively decreases bone turnover in a dose-related manner and increases lumbar BMD at a dosage of 2.5mg/day, the lowest dose used in this study, in Japanese patients with osteoporosis.

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The high incidence of childhood thyroid cancer in Belarus is suspected to be due to radiation exposure after the Chernobyl reactor accident. To clarify the clinical and histological characteristics of childhood thyroid cancer in Belarus, we therefore compared these patients to a radiation non-exposed control series in Japan. In Belarus, 26 thyroid cancers in subjects aged 15 or younger were diagnosed among 25, 000 screened between 1991 and 1995 by Chernobyl-Sasakawa Health and Medical Cooperation Project. The clinical and morphologic features of these 26 cases were compared to 37 childhood thyroid cancers in Japan diagnosed between 1962 and 1995. The age distribution at operation in Belarus showed a peak at 10 years old, with a subsequent fall in numbers. In contrast, the age distribution at operation in Japan showed a smooth increase between the ages of 8 and 14. The mean tumor diameter was smaller in Belarus than that in Japan (1.4±0.7 vs. 4.1±1.7cm, P<0.001). The sex ratio, regional lymph node metastasis, extension to surrounding tissues or lung metastasis did not differ significantly. Histologically, all cases in Belarus were papillary and in Japan 33 cases were papillary and 4 cases were follicular carcinomas. Among papillary carcinomas, the frequency of a solid growth pattern, a criteria for classifying a tumor as poorly differentiated, was higher in Belarus than that in Japan (61.5 vs.18.2%, P<0.001). The difference between the features of childhood thyroid cancer in Japan and Belarus may be due to the difference in the process of carcinogenesis, but more direct evidence and further analysis by molecular epidemiology are needed in Belarussian cases.

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A 38-yr-old female with a TSH- and GH-secreting pituitary adenoma is described, who had both overt symptoms, hyperthyroidism and acromegaly. Her serum TSH was not suppressed despite high concentrations of free T3 and free T4, and her α-subunit/TSH molar ratio was high. Her serum GH was consistently high, and was not suppressed by an oral glucose tolerance test. Preoperative testing revealed that, although the TSH response was impaired, TSH, α-subunit and GH were increased by TRH injection, and that these hormones were reduced by bromocriptine or somatostatin analog. Although she did not have hyperprolactinemia, the in vitro culture and immunohistochemical studies revealed that the adenoma cells produced and released PRL, in addition to TSH, α-subunit and GH. Immunohistochemical studies showed the presence of GH in the cytoplasm of many adenoma cells. TSHβ-positive adenoma cells were less frequently seen than GH-positive adenoma cells. No cells showed the coexistence of GH and TSHβ, and a few cells were positive for PRL. By electron microscopy, the adenoma was found to be composed of a single cell type resembling thyrotrophs, and did not have any characteristics of somatotrophs. This case was considered to be of interest, because the adenoma was ultrastructurally monomorphous, but immunohistochemically polymorphous.

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Children with simple obesity (SO) show increased linear growth with normal or high serum insulin-like growth factor-I (IGF-I) levels during prepubertal period, despite low GH secretion. We measured IGF-I, IGFBP-1, GHBP and other factors to clarify the hormonal relation between the nutrition and the linear growth in SO and compared these factors with children with normal short stature (NS). Subjects were 23 SO and 19 NS children, and their height standard deviation (SD) scores were 0.7±0.2 SD and -3.4±0.3 SD (mean±SEM) (P<0.01), respectively. Oral glucose tolerance test (OGTT) was performed in all the subjects and GH-releasing factor (GRF) test was also performed in 13 of SO and 17 of NS. The peak levels of GH in the GRF test were significantly lower in SO than in NS (12.8±1.7 vs. 39.8±6.9ng/ml) and showed a significantly positive correlation with ∑IGFBP-1 (r=0.63, P<0.01). Serum GHBP level and IGF-I level were significantly higher in SO than in NS on pubertal stage matching. There was a positive correlation between GHBP and ∑insulin during OGTT (r=0.75, P<0.01). When the sum of the values during OGTT was expressed as ∑, ∑insulin, ∑C-peptide and glucose were significantly higher in SO than in NS on pubertal stage matching. Basal and ∑IGFBP-1 were significantly lower in SO than in NS, but IGFBP-3 levels showed no significant difference between the two groups either in prepuberty or midpuberty. In conclusion, it can be hypothesized that the overnutrition causes hyperinsulinemia which increases GH receptor and IGF-I secretion despite low GH secretion. Hyperinsulinemia also may increase free IGF-I by lowering IGFBP-1. These two mechanism are supposed to be the nutrition related hormonal changes in SO and can explain the growth of SO. In addition, the increased free IGF-I may contribute the decreased GH secretion due to negative feedback in SO.

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The effects of carboxyl-terminal (C-) PTH fragments, (35-84), (53-84) and (69-84), on the proliferation and function of osteoblastic UMR-106 cells were compared with those of amino-terminal (N-) (1-34) and intact (I-) (1-84) PTH. I-PTH as well as N-PTH at 10^-8M significantly inhibited [³H] thymidine incorporation and stimulated alkaline phosphatase activity in UMR-106 cells. No C-PTH fragments affected them. In contrast, the expression of type-1 procollagen mRNA in these cells was stimulated by all C-PTH fragments, inhibited by N-PTH and not affected by I-PTH. All C-PTH fragments except (69-84) as well as N-PTH and I-PTH stimulated IGFBP-5 mRNA expression. The present study suggests that the C-portion of the PTH molecule exercises biological activities in mRNA expression of type-1 procollagen as well as IGFBP-5 in osteoblasts, and that it might be involved in the anabolic action of PTH on bone in vivo.

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Androgen has a potency to induce female sexual behavior in ovariectomized rats. This study investigated the inhibitory effect of progesterone (P) on lordosis behavior facilitated by androgen. Ovariectomized female rats were given 100 to 800μg/kg testosterone propionate (TP) or 5μg/kg estradiol benzoate (EB). Forty-four hours after administration of the TP or the EB, all females received 0.5mg P, and the first behavioral test was carried out four hours later. TP-treated animals showed lordosis responses in a dose dependent manner. Two weeks after the first test, the animals were retested by using the same hormonal regime as in the first test, except that an additional 5mg P was administered 1h prior to the TP or the EB. As a result, significant reductions in sexual receptivity were observed in the TP-treated as well as in the EB-treated females. These results indicate that progesterone can inhibit androgen-induced lordosis behavior in female rats.

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The present study was undertaken to determine plasma adrenomedullin levels in patients with non-insulin dependent diabetes mellitus (NIDDM) to elucidate the potential involvement in the pathogenesis of diabetic complications. The patients were 24 males and 21 females with ages of 55±2.1 years (mean±SEM). Plasma adrenomedullin levels were 5.94±0.44pmol/l in patients with NIDDM, and were not affected by plasma glucose concentration. The plasma adrenomedullin increased dependent on the severity of diabetic nephropathy and retinopathy. Plasma levels of adrenomedullin positively correlated with various parameters, including serum creatinine levels, urinary excretion of protein, and systolic blood pressure. In contrast, there were negative correlations between the coefficient variation (CV) of RR intervals and plasma adrenomedullin, and between the conduction velocity of ulnar nerves and plasma adrenomedullin levels. These results indicate that the increase in plasma adrenomedullin was closely related to diabetic complications, which may be dependent on the development of microangiopathy.

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Although serum deprivation induces apoptosis in several cell lines, biochemical characterization of the apoptosis in primary granulosa cells (GCs) induced by serum deprivation has rarely been reported. In the present study, GCs from small follicles of porcine ovaries were precultured under a serum-containing condition for seven days, then stepped down to a serum-free condition and cultured for the subsequent two days. GCs were subjected to DNA fragmentation and immunoblot analyses. Data indicated that serum deprivation induced GC apoptosis characterized by DNA laddering, which was associated with decreased expression of proliferating cell nuclear antigen (PCNA) and increased expression of p53 protein, Fas antigen and Fas ligand. Serum deprivation also resulted in an increase in a 115kDa protein expression despite no detectable expression of a 66kDa c-myc protein. This suggests that serum removal from primary GCs may activate multiple apoptotic pathways such as a p53-associated pathway and a Fas-Fas ligand pathway.

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We recently reported three cases of severe hypertriglyceridemia caused by tamoxifen-treatment after breast cancer surgery. In that report we showed that one of the three patients had apo E3/3 and another had E4/2 phenotype. There are few data about the relationship between apoprotein E phenotype, one of modifiers of lipoprotein metabolism, and tamoxifen induced lipemia. In the present study, we studied apo E phenotype to clarify the relationship between apo E phenotype and the changes in lipids during tamoxifen treatment. Plasma triglyceride levels in apo E3/3 and apo E4/3 were 114±9 mg/dl and 87±12mg/dl, respectively, before tamoxifen treatment, and increased significantly to 191± 35mg/dl in apo E3/3 and 167±35mg/dl in apo E4/3 during tamoxifen treatment. There was no significant difference between apo E3/3 and apo E4/3 in triglyceride levels before and during tamoxifen treatment. Plasma levels of total cholesterol during tamoxifen treatment in both apo E3/3 and apo E4/3 were similar to those before treatment. Our data suggest that the increase in triglyceride during tamoxifen treatment may occur in every type of apo E phenotype.

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A 45-year-old Japanese woman, treated for Bartter's syndrome for 14 years, presented with complaints of numbness in her extremities and polyarthralgia. She was diagnosed to have Gitelman's syndrome with chondrocalcinosis, which were effectively treated with spironolactone and magnesium supplementation. Gitelman's syndrome is a primary renal tubular disorder characterized by hypomagnesemia and hypocalciuria with normal calcemia. The persistent hypomagnesemia is one of the causes of chondrocalcinosis, and many cases of Bartter's syndrome with hypomagnesemia are associated with chondrocalcinosis attributed to a tubular magnesium defect. We summarize the reported cases with Bartter's syndrome and chondrocalcinosis, referring to the possibility of Gitelman's syndrome.

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Preoperative therapy with octreotide, a long-acting somatostatin analog, suppresses GH hypersecretion, shrinks GH-producing tumors and leads to an improvement in subsequent surgical remission in acromegalic patients. A continuous infusion of octreotide has demonstrated more persistent suppression of GH secretion than intermittent injections, and only a few studies were reported on the effect of the tumor shrinkage with a continuous infusion of a small dose of octreotide. We therefore investigated the preoperative effects of small doses of octreotide (120-240μg/day) administered continuously (with a subcutaneous infusion pump) over a short period (2 or 4 weeks) in nine untreated acromegalic patients. Octreotide therapy resulted in suppression of serum GH and IGF-1 concentrations in 8 out of 9 patients and reduction in pituitary tumor size measured by MRI in all patients (by 7.9 to 38.5%). In particular, considerable reduction in tumor size (more than 20%) occurred in 6 of 9 patients. In three patients assessed serially throughout the preoperative period, reduction in tumor size was noted within only one week after the start of octreotide therapy and reduction rate more than 20% was obtained within the first two weeks. In one patient, suprasellar tumor expansion totally disappeared after such therapy. Our results indicate that short-term continuous subcutaneous infusion of a small dose of octreotide results in not only inhibition of GH hypersecretion but also shrinkage of tumor size prior to surgery.

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The relationship between magnesium levels and oxygen uptake in patients with non-insulin dependent diabetes mellitus (NIDDM) without apparent visceral dysfunction was studied. Magnesium levels in plasma, erythrocytes and urine as well as oxygen uptake parameters were determined in NIDDM patients and controls. Oxygen uptake parameters were measured with an exercise ergometer and expired gas analysis according to Wasserman et al. Low oxygen uptake in NIDDM patients was correlated significantly with plasma and erythrocyte magnesium levels, but not with urinary magnesium excretion. In NIDDM patients, higher correlation coefficients were seen between oxygen uptake parameters at peak in men and at the anaerobic threshold in women and plasma or erythrocyte magnesium levels. These results confirm previous results indicating that plasma and erythrocyte magnesium levels in NIDDM patients are decreased, and further demonstrate that the decreased magnesium levels are positively correlated with oxygen uptake. Although the mechanism remains to be established, it is possible that the magnesium deficiency in NIDDM patients due to environmental or genetic factors may result in low oxygen uptake and decreased work capacity.

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