Chronic hyperglycemia causes insulin resistance, termed glucose toxicity. Herein we studied chronic glucose-dependent regulation of the glucose transport system in adipocytes. 3T3-L1 adipocytes were incubated for up to 24h with low (1mM) or high (25mM) glucose, and glucose transport was subsequently analyzed. 100nM insulin was present throughout the experiments. 24h incubation with 1mM glucose caused a 2.3±0.4 fold increase in glucose transport activity, compared to the values obtained with 25mM glucose. This difference was not observed when 24h incubation was carried out without insulin. Glucose transport activity was not increased at 3 or 6h incubation with 1mM glucose, but was increased at 12h, which closely paralleled increased expression of GLUT1. In addition to increased GLUT1 expression, more efficient translocation of GLUT1 to the plasma membrane was observed when incubated with 1mM glucose compared to 25mM glucose. The addition of azaserin or deprivation of glutamine at 25mM glucose did not increase the glucose transport activity to the level obtained with 1mM glucose. PD98059 did not affect glucose transport activity when incubated with 1mM or 25mM glucose. In conclusion, the present study is the first to show that, in 3T3-L1 adipocytes, chronic exposure to low (1mM) and high (25mM) glucose leads to different insulin-stimulated glucose transport activities. These differences result from the difference in the expression and plasma membrane distribution of GLUT1, but not of GLUT4, and the hexosamine biosynthesis pathway or extracellular signal-regulated protein kinase is not involved.

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We recently isolated a mammary growth factor from mouse mammary fibroblast-conditioned medium and identified it as a homologue of human HGF (hepatocyte growth factor) (Sasaki et al. Biochem Biophys Res Commun 199: 772-779, 1994). To elucidate the role of mammary fibroblast-derived HGF in mammary epithelial cell growth in vivo, we used recombinant mouse HGF and examined its effect on the growth of primary cultures of mouse mammary epithelial cells in this study. HGF stimulated the growth of mammary epithelial cells not only in monolayer culture but also in three-dimensional collagen gel matrix culture. In contrast, mammogenic hormones, i.e., prolactin and progesterone, stimulated the growth of mammary epithelial cells only when the cells were cultured in a collagen gel matrix, suggesting that the three-dimensional culture conditions are necessary to observe the growth-stimulatory effect of mammogenic hormones seen in vivo. Under these three-dimensional culture conditions, an additive effect of HGF and mammogenic hormones was also seen. Northern blot analysis revealed that HGF mRNA was expressed in the mammary gland as well as in cultured mammary fibroblasts but not in cultured mammary epithelial cells, indicating that HGF is produced preferentially by the stromal fibroblasts of the mammary gland. These observations thus suggest a role for HGF as a mammary stromal fibroblast-derived growth factor which, in cooperation with mammogenic hormones, stimulates the growth of mammary epithelial cells in vivo.

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To examine the mechanism behind a decrease in type 1 iodothyronine deiodinase (D1) gene expression in diabetes mellitus, we evaluated the effect of administering T3 and/or insulin on D1 activity and the mRNA levels in the liver of streptozotocin (STZ)-induced diabetic rats. STZ (100mg/kg BW) was administered to male Wistar rats, and the rats were divided into four groups as follows: (1) STZ alone, (2) STZ and T3 (5μg/100g BW daily for 7 days), (3) STZ and insulin (intermediate-acting insulin, 4units/100g BW daily for 7 days), and (4) STZ, T3, and insulin. Blood glucose levels increased in Group 1, but were normalized in Group 3. Serum T3 levels were markedly decreased in Group 1. They were within normal limits 24hours after the last administration of T3 in Group 2 and after the administration of insulin in Group 3. T3 levels were supranormal in Group 4. TSH levels were normal in Groups 1 and 3, but were suppressed in Groups 2 and 4, suggesting that rats in Groups 2 and 4 were actually in a hyperthyroid state after injecting a large amount of T3. D1 activity in Group 1 was reduced significantly, but it was normal in Groups 2 and 3, and increased in Group 4. D1 mRNA levels in the liver in Group 1 decreased significantly, but they were increased to within normal limits by adding insulin in Group 3. They were also normal in Group 2 where hyperglycemia was evident and rats were hyperthyroid after administering T3. D1 mRNA in Group 4 increased significantly where glucose levels were normal and T3 levels were increased. We suggest that the decrease in hepatic D1 mRNA in STZ-induced diabetic rats is due to metabolic derangement caused by insulin deficiency in addition to a possible decrease in tissue T3 availability.

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We previously reported the inhibition of cell-growth in Neuro-2A cells, mouse neuroblastoma, by Zn²⁺ chelation with EDTA. This paper describes the purification of a factor that prevents EDTA-induced cell-growth inhibition from chick embryo brain. The purified factor has a molecular mass of 16kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions. This factor prevents the cell-growth inhibition in a dose-dependent manner and also binds thyroxine. Analysis of the N-terminal amino acid sequence revealed that 40 residues coincide with the sequence of chicken liver transthyretin.

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Interferon-tau (IFNτ) is produced by the trophectoderm of ruminant ungulates and its gene transactivation in vitro has so far been achieved only in human choriocarcinoma cells, JAR and JEG3. To examine if ovine IFNτ gene transactivation could be induced in cells other than JAR or JEG3 cells and its activation could be aided by the expression of a protooncogene(s), a transient transfection system was developed with the upstream region of ovine IFNτ gene that had been inserted into the chloramphenicol acetyltransferase (CAT) reporter plasmid (IFNτ-CAT). The effect of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), on IFNτ-CAT transcriptional activity was examined in JEG3, human embryonic kidney (293), HeLa and Vero cells. Upon transfection and PMA treatment, ovine IFNτ gene was transactivated in two unrelated cell lines, JEG3 and 293 cells. Since IFNτ-CAT was not induced in HeLa or Vero cells, HeLa and JEG3 cells were further examined for their ability to support IFNτ-CAT transactivation in a co-transfection system. While the expression of c-myc, interferon regulatory factor 1 or 2 (IRF-1 or IRF-2) was not effective, CAT activity was strongly enhanced in both JEG3 and HeLa cells with the co-transfection of c-Jun or c-Jun plus c-Fos. These data suggest that ovine IFNτ gene transcription induced by PMA is not specific for trophoblast cells and a protooncogene, c-jun, is a downstream effector of PMA activated nuclear factors in its signal transduction cascade resulting in IFNτ gene transactivaion.

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A rat pituitary tumor sub-line MtT/E-2 was established from a rat pituitary tumor cell line MtT/E. Its growth was found to depend on the presence of estradiol (E₂) in culture media at 10^-13-10^-9M, whereas the original cell line MtT/E proliferated autonomously. The recently discovered PTTG (pituitary tumor transforming gene) is highly expressed in this cell line, although not regulated by E₂. On the other hand, E₂ induced c-myc and cyclin D1 proteins in MtT/E-2, which contains a lot (220±18fmol/mg protein) of estrogen receptor demonstrated by RT-PCR analysis to be predominantly α type. MtT/E-2 secretes growth hormone which is, interestingly, regulated by retinoic acid and dexamethasone rather than thyroid hormones.

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The Inui and Ochi group recently reported that CAMP production by porcine thyroid cells (PTC) was augmented more by polyethylene glycol (PEG) 22.5% precipitated fractions from almost all Graves' sera than those of PEG 12.5%. In the present study, thyroid stimulating immunoglobulin (TSI) activity was determined with PTC and prepared crude Ig fractions precipitated by two different concentrations of PEG (final concentrations 13.5% and 22.5%) from sera obtained from 117 Graves' patients. The activity of TSI determined by the PEG 13.5% assay and activity determined by the PEG 22.5% assay were designated as thyroid-stimulating antibody (TSAb) and sTSAb, respectively. At first we studied 55 TSAb-positive patients with untreated hyperthyroid Graves' disease and classified them according to the TSAb activity-below 500% (group 1) and above 500% (group 2). The positive stimulatory effect, arbitrarily defined as the ratio of sTSAb to TSAb, being more than 1.2, was observed in 85% of patients, and group 1 had a significantly (P<0.025) greater stimulatory effect (34/35, 97.1%) than group 2 (13/20, 65%). Subsequently, in 29 TSAb-negative patients, sTSAb was measured and detected in 26 (89.7%). Finally, sTSAb, TSAb and TBII were compared between patients presenting with recurrent Graves' disease and those with silent thyroiditis after withdrawal of antithyroid drug treatment for Graves' disease. sTSAb was detected in all 14 relapsed patients, but none of the 9 patients with silent thyroiditis had detectable sTSAb. In contrast, TSAb and TBII activities were found in only 7 (50.0%) of the 14 relapsed cases. The present paper demonstrated that the assay with a higher PEG concentration was found to be sensitive, specific and useful for the diagnosis and follow-up of Graves' disease after drug withdrawal, although the underlying mechanism remains unclear.

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It has been suggested that prostate homeostasis is regulated indirectly by androgens through stromal-epithelial interactions in part by factors from the stromal cells acting on receptors in epithelial cells. In this report, the role of fibroblast growth factor (FGF)-10 in prostatic epithelial proliferation was investigated. The expression of FGF-10mRNA was apparent in primary-cultured stromal cells, but not in epithelial cells derived from human tissue from patients with benign prostatic hyperplasia (BPH). The mitogenic activity of human recombinant FGF-10 assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation was demonstrated in isolated epithelial cells, but not in cultured stromal cells. No mitogenic activity of dihydrotestosterone (DHT) for either epithelial or stromal cells could be demonstrated, but quantitative PCR (real-time PCR) with a double-labeled fluorogenic probe demonstrated that expression of FGF-10 in stromal cells was enhanced 5.3-fold at a DHT concentration of 100pM. Androgen receptor mRNA levels showed no significant change with DHT at concentrations less than 100pM, but were reduced to 50% of control levels at a DHT concentration of 10nM. These results suggest that stromal-derived FGF-10 stimulates human prostatic epithelial growth and its mRNA expression is induced by androgens, without an increase in the androgen receptor mRNA. Moreover, FGF-10 may be involved in the development or support of human BPH.

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The gene for human growth hormone receptor (hGHR) consists of at least 10 exons, and the corresponding protein is encoded in exons 2-10 which span at least 87kbp of chromosome 5. Failure to amplify exons 3 and 8 of the hGHR gene from Japanese subjects with the previously reported primers prompted us to determine intron sequences flanking exon 3 and those flanking exon 8 of the hGHR gene, and novel intron sequences flanking exons 3 and 8 of the hGHR gene were identified. We designed new oligonucleotide primers based on these sequences, and successfully amplified DNA fragments encompassing exon 3 and those encompassing exon 8 of the hGHR gene. Since all of the 50 Japanese and the two Caucasians had the very same intron sequences which were different from the previously reported ones, it is more likely that the previously reported sequences were simply wrong than that there exist polymorphic differences in the intron sequences among different ethnic populations.

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Nitric oxide (NO) has divergent actions under physiological and pathological conditions. NO is rapidly decomposed to nitrite (NO₂^-) and nitrate (NO₃^-). Since these metabolites are stable, they are good indices of NO production under various conditions. In the present study, we measured NO₂^- and NO₃^- concentrations in the urine collected from 62 hospital controls and 504 healthy subjects by means of a new HPLC system combined with Griess reaction. NOx was the sum of NO₂^- and NO₃^- There was no considerable inter-day variation in urinary NO metabolite levels, and there was close correlation between NO₂^-, NO₃^- and NOx values in spot urine obtained in the early morning and those in 24-h stored urine in hospital controls. Urinary NO metabolite levels, which were corrected by creatinine (Cr) excretion and expressed on a logarithmic scale, showed normal distribution and were independent of sex and age in healthy subjects. The normal ranges of urinary NO₂^-, NO₃^- and NOx levels were estimated as 17-72μmol/g Cr, 1, 023-2, 818μmol/g Cr, and 1, 071-2, 951μmol/g Cr, respectively. We also found that urinary NO metabolite levels were lower than normal range in patients with various diseases.

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We report a case of giant cystic insulinoma constituting part of multiple endocrine neoplasia (MEN) type I. A 29-year-old Japanese man presented with a history of recurrent hypoglycemic attacks. Endocrine examination showed hyperinsulinemia discordant with hypoglycemia, and a giant cystic insulinoma (11×10cm) located in the pancreatic tail was detected radiologically. Hyperprolactinemia due to pituitary adenoma and hyperparathyroidism due to parathyroid hyperplasia were also present. The insulinoma, prolactinoma and hyperplastic parathyroid gland were surgically removed. Fluorescent microsatellite analysis detected loss of heterozygosity (LOH) in chromosome 11q13 in DNA samples from all resected tissues but not from white blood cells. This is a rare case of MEN type I because of the giant cystic insulinoma and the evidence of common LOH detected in all MEN type I tissues.

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The effect of thyroid hormone on immune function is unclear. The influence of L-triiodothyronine on expression of the interleukin-2 receptor alpha chain by peripheral blood mononuclear cells from healthy volunteers and YT cells (an interleukin-2 independent natural killer-like cell line) was examined. Concanavalin A stimulation significantly (p<0.05 and p<0.01) increased soluble interleukin-2 receptor alpha chain production when mononuclear cells were cultured with triiodothyronine (1-100nmol/l) for 3days. The stimulatory effect of triiodothyronine on interleukin-2 receptor alpha chain expression was greater in the presence of concanavalin A (5μg/ml) plus interleukin-2 (1U/ml) than in the presence of concanavalin A alone. Triiodothyronine also significantly (p<0.01) increased interleukin-2 receptor alpha chain expression when YT cells were cultured for 2 days with interleukin-2 (1U/ml), but did not influence receptor expression when YT cells were cultured with forskolin or 12-O-tetradecanoyl phorbol 13-acetate, potent activators of signal transduction. In conclusion, triiodothyronine may have an immunomodulatory effect by enhancing expression of the interleukin-2 receptor alpha chain on peripheral blood mononuclear cells in the presence of interleukin-2.

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Since amiodarone was introduced in Japan in 1992, the incidence of the drug-induced thyroid dysfunction has been increasing. We studied the thyroid function of 13 patients with amiodarone-induced thyrotoxicosis (AIT) and 11 patients with amiodarone-associated hypothyroidism (AAH) who had been referred to our Institute in the last 6years. AIT and AAH developed after 39±21 and 20±16 months of amiodarone treatment, respectively. One patient developed AAH followed by AIT. The AIT ranged from subclinical to overt thyrotoxicosis. Four patients with moderate to marked AIT were treated with methimazole. Their thyrotoxicosis persisted for 3 to 9months, despite administration of antithyroid agents. One patient with mild thyrotoxicosis was treated with prednisolone, resulting in a euthyroid state in a few months. Eight patients with asymptomatic to moderate thyrotoxicosis resolved spontaneously without any treatment. In four asymptomatic patients with AIT, serum levels of T₃ and T₄ were in the upper normal range or slightly high (<12μg/dl), accompanied by suppressed TSH (<0.1 μU/ml) and high thyroglobulin levels, suggesting destruction-induced thyrotoxicosis. Such a subclinical thyrotoxicosis developed repeatedly in one patient. Ultrasonographic studies revealed no nodular lesion in the thyroid, and color flow Doppler sonography demonstrated no hypervascularity in the thyroid gland in any AIT patient. Although it is postulated in Europe that there are two types of AIT, namely type I, which develops in patients with latent Graves' disease or toxic multinodular goiter, and type II, which develops in an apparently normal thyroid as destructive thyroiditis, all AIT patients we have seen so far had developed destructive type AIT. Sufficient intake of iodide and a very low incidence of toxic multinodular goiter may account for the rare incidence of type I AIT in our country. Mild to moderate AIT resolved spontaneously without discontinuing amiodarone, but it was discontinuedin two of 13 AIT patients because of extrathyroidal adverse reactions.

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2-Hydroxyestradiol 17-sulphate (2-OH E₂-17-S) is a catecholized form of sulphated estrogen. In vitro studies showed that its antioxidative effect is almost equal to that of free catecholestrogens, such as 2-OH E₂ or 4-OH E₂ and α-tocoferol, but the existance of 2-OH E₂-17-S in human serum has not yet been made clear. 2-OH E₂-17-S strongly antagonizes lipid peroxidation, and so it may play an important role in pregnancy, for example as an anti-oxidant in pregnancy-induced hypertension (PIH). The serum level of 2-OH E₂-17-S was measured during mid to late pregnancy by a direct radioimmunoassay (RIA) without hydrolysis. The serum levels at 28-31 weeks, 32-35 weeks and 36-40 weeks of gestation were 4.68±0.93 (mean±SE), 8.38±1.21 and 18.31±3.41nmol/l, respectively. The serum level in PIH cases at 36-40 weeks (4.64±1.29nmol/l) was significantly lower than that in normal pregnancy. The 2-OH E₂-17-S level in umbilical arteries was significantly higher than that in maternal peripheral vein. These results suggest that the feto-placental unit plays an important role in catecholizing E₂-17-S to 2-OH E₂-17-S, which may act as an antioxidant in pregnancy.

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To examine the specific changes in gene expression in thyroid carcinomas, we performed sequence specific-differential display (SS-DD) analysis by using a specific degenerate primer for the superfamily of the small G protein. After subcloning and sequencing analysis, one of the genes that showed decreased expression in thyroid papillary carcinomas was revealed to be the proto-oncogene c-fos. Expression of c-fos mRNA in benign and malignant tissues was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. The expression of c-fos was detected in all twelve normal thyroid tissues, whereas it decreased in thirteen of fifteen papillary carcinomas. These results indicate that the increased expression of c-fos mRNA is not necessarily to be an oncogenic feature of thyroid tumors. Further, its constant expression in normal thyroid tissues may play a role in the maintenance of thyroid function.

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Vitamin D receptor gene polymorphism was determined in 66 and 60 Japanese patients with prostate cancer and non cancer controls, respectively. In contrast to previous reports showing an association between vitamin D receptor polymorphism of a TaqI restriction fragment length polymorphism at codon 352 (genotype tt) and prostate cancer in an American population, the frequency of genotype tt is less than one percent in the Japanese population. There was no difference between the patients and the controls in the vitamin D receptor TaqI genotype. In patients with metastatic prostate cancer, genotype TT had a tendency to shorter progression free survival compared to genotype Tt, but the number of patients was limited

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We have shown that dexamethasone stimulates insulin-like growth factor binding protein-1 (IGFBP-1) production due to an increase in IGFBP-1 mRNA content in primary cultures of rat hepatocytes. In the present study, we investigated the effect of dexamethasone on the stabilization of IGFBP-1 mRNA in this system. We found that dexamethasone stabilized IGFBP-1 mRNA from the dot blot analysis with actinomycin D in the culture medium. It is suggested that the stabilization of IGFBP-1 mRNA is one of the mechanisms in which IGFBP-1 mRNA increases during dexamethasone treatment in primary cultures of rat hepatocytes.

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