In the present study, the effects of various cytokines on parathyroid hormone-related protein (PTH-rP) production and PTH-rP mRNA expression in human amnion cells were studied. Immunoreactive (ir) PTH-rP was measured by immunoradiometric assay and the expression of PTH-rP mRNA was determined by Northern blot analysis. The addition of interleukin-1β (IL-1β, 10ng/ml) and IL-6 (10ng/ml) in culture medium for 24 hours resulted in a significant increase in ir-PTH-rP levels by 1.5 and 1.6 fold, respectively. The effects of these agents were dose dependent. In contrast, IL-2 (10ng/ml) and IL-8 (10ng/ml) showed no effect on the production of ir-PTH-rP from amnion cells. Treatment with IL-1β or IL-6 for 6 hours increased the expression of PTH-rP mRNA in amnion cells. The stimulatory effect of IL-1β was reduced by IL-1 receptor antagonist (IL-1Ra) in a dose dependent manner. Both tetradecanoyl phorbol acetate (TPA), and forskolin increased PTH-rP mRNA levels and the PTH-rP production in amnion cells, and the effect of TPA was much greater than that of forskolin. The findings of the present study suggest of the participation of inflammatory cytokines for the regulation of PTH-rP production in human amnion cells.

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Lipopolysaccharide (LPS) is known to stimulate the synthesis and secretion of various proinflammatory cytokines in both the peripheral immune cells and the brain. Yet, the relative contribution of peripheral and central cytokines to the LPS-induced activation of the hypothalamo-pituitary-adrenal axis is still poorly understood. In this study, utilizing the push-pull perfusion technique of the rat brain, we attempted to characterize in detail the temporal profiles of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α after intravenous (iv) or intraperitoneal (ip) administration of LPS in both the general circulation and the hypothalamic paraventricular nucleus (PVN), which is the primary source of corticotropin releasing hormone (CRH). Temporal changes in plasma adrenocorticotropic hormone (ACTH) and CRH levels in the PVN were also monitored. We collected blood and perfusates every 30min from 11:00 to 17:00h. At 12:00h, 1.0 or 2.5mg/kg body weight of LPS was given ia an iv or ip route, respectively. Peak ACTH response occurred 30min after iv LPS and 1.5h after ip LPS. Of the three cytokines measured in the plasma, TNF-α showed the fastest rise in synchrony with peak ACTH secretion after both iv and ip LPS. Although plasma IL-6 also showed a robust rise, its peak level occurred later than the ACTH peak. Elevation of plasma IL-1β was the smallest among the three cytokines. CRH levels in the PVN reached their peaks 1 and 2.5 h after the ACTH peak following ip and iv LPS, respectively. Irrespective of the route of LPS administration, IL-6 and TNF-α levels in the PVN showed significant rises 1-2h after the ACTH peak, but IL-1 in the PVN did not significantly change during the entire period of observation. The results of the present study suggest that circulating TNF-α may play the most important role in triggering the early, peak phase of ACTH secretion after both iv and ip LPS. Although it is possible that brain TNF-α, IL-6, and circulating IL-6, may be involved in the later, protracted phase of ACTH secretion induced by LPS, IL-1β in both the brain and peripheral circulation seems to play the smallest role in ACTH secretion. This is the first study to characterize the LPS-induced temporal changes in IL-1β, IL-6, and TNF-α in both plasma and PVN simultaneously in conscious, freely moving rats.

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We investigated the effects of streptozotocin (STZ)-induced diabetes on thyroid hormone levels, type 1 deiodinase (D1) activity and messenger RNA (mRNA) levels in inherited D1 deficient C3H mice in a comparative manner with control C57 mice. The apparent maximum velocity (V_max) D1 values in C3H mice were 3% (liver) and 26% (kidney) of those in C57 mice. In C3H mice, similar serum T3, slightly higher T4, and 2.6-fold higher rT3 levels were observed compared with C57 mice. In STZ-induced diabetes, serum T4 level markedly decreased in both C3H and C57 mice. Serum T3 levels in STZ-C3H mice similarly decreased as in STZ-C57 mice. On the other hand, serum rT3 levels increased to 3.3-fold higher in STZ-C3H than in STZ-C57 mice. The Vmax values were decreased to 12% (STZ-C3H) and to 30% (STZ-C57) in liver, and decreased to 33% (both STZ-C3H and STZ-C57) in kidney. The changes in D1 mRNA levels in diabetes versus control were comparable to those of D1 activities in both strains. In summary, similar mechanism(s) to those which decrease the D1 expression and the serum T3 level in diabetes, function in D1 deficient C3H mice as in C57 mice. It appears that hepatic and renal D1 activity alone can not explain the similar reduction in T3 level in STZ-C3H mice and STZ-C57 mice.

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This report describes the clinical and pathological characteristics of two patients with lymphocytic hypophysitis (LHy) and two with infundibuloneurohypophysitis (INHy). Two of the patients were women and two were men, and their ages were between 27 and 38 years old. This disease was not associated with either pregnancy or the postpartum period in the female patients. Two of the patients presented with diabetes insipidus, one with panhypopituitarism and right abducens paralysis and one with headache and galactorrhea. At presentation three of the patients had mild to moderate hyperprolactinemia and one had low prolactin levels. All four had abnormal magnetic resonance imaging (MRI): focal nodular enlarging of the infundibulum and normal hypophysis in one, expanding sellar masses in two, and diffusely thickened stalk with slightly enlarged pituitary gland in one. Three cases showed no sign of adenohypophysial deficiency with stimulation tests. One patient had associated chronic lymphocytic thyroiditis. Of the first three patients, one patient underwent transcranial and two underwent transnasal transsphenoidal (TNTS) surgery for mass excisions since they were thought to have pituitary tumors. Endoscopic endonasal transsphenoidal biopsy was performed in the last one with a suspicion of LHy. The pathological and immunohistochemical examinations revealed lymphocytic infiltration. Hyperprolactinemia resolved with surgery in two patients and one developed diabetes insipidus as a complication. We conclude that LHy and infundibuloneurohypophysitis should be considered in the differential diagnosis of the mass lesions of the sellar region and also should be kept in the mind for the etiopathogenesis of cases of hyperprolactinemia, galactorrhea and diabetes insipidus. In suspected cases endoscopic endonasal biopsy for the histopathological diagnosis can be a safe approach.

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Endocrine disruptors are a diverse group of chemicals that alter the functions of the endocrine system. A large proportion of endocrine disruptors have estrogenic effects, thus are called environmental estrogens. In the present study, an estrogen (E₂) responsive rat pituitary cell line, MtT/E-2, was employed to examine 1) the potency of several endocrine disruptors including bisphenol A (BPA), o, p'-DDD, methoxychlor, 1, 2, 3, 4, 5, 6-hexachlorocyclohexane (HCH) and dibromoacetic acid (DBAA) in terms of E₂ responsive pituitary cell growth; 2) whether BPA has estrogenic action in vivo causing the growth of MtT/E-2 cells grafted in rats. Binding assays showed the test chemicals were able to compete with ³H-E₂ binding to the estrogen receptor (ER). The compounds also stimulated growth of MtT/E-2 cells at rates corresponding to their ER binding affinity. Their transcription activation of an (ERE)₃-SV40-luciferase reporter in MtT/E-2 cells was comparable to their stimulation of cell growth, with the exception of HCH which showed little induction of cell growth but strong stimulation in ERE dependent transcription activation. MtT/E-2 cells were inoculated into ovariectomized female F344 rats treated with E₂ or BPA. The first tumors were noted at day 22 in the E₂ treated group, at day 25 in the highest dose of BPA group and at day 41 in the control group. These results suggest 1) that the growth assay with MtT/E-2 cells provides simple and sensitive test for detection of estrogenic activity of environmental chemicals; 2) that BPA has estrogenic potency to stimulate E₂ responsive cell growth in vivo as well as in vitro.

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Immature rats receiving equine chorionic gonadotropin (eCG) and human CG (hCG) were used to study the time course changes in nitric oxide synthase (NOS) activity in the ovary during ovulation. To study the role of NO in ovulation, the effects of intrabursal injection of L-NG-monomethylarginine (L-NMMA, 125μg/20μl/bursa), a NOS inhibitor, on the number of ova shed were also examined. Rats were sacrificed at -48, 0, 3, 6, 9, 12, and 24 h after hCG injection, and the ovaries were collected for the NOS activity assay, Western blotting, NADPH-diaphorase histochemistry and immunohistochemistry. Total NOS and constitutive NOS activities in the ovary increased significantly at 9h after hCG injection and the values remained high thereafter. Inducible NOS (iNOS) activity was detectable as a small peak at 3 and 6h after hCG injection. Endothelial NOS (eNOS) protein production increased after hCG injection with a peak at 12h, whereas iNOS protein production decreased at 12 and 24h after hCG injection. NADPH-diaphorase positive cells increased at the thecae of growing follicles after hCG injection, appeared at mural granulosa cells before ovulation, and were detected in newly formed corpora lutea, which coincided with the results in eNOS positive cells by immunohistochemistry. L-NMMA given to rats at 5 or 7h after hCG was most effective in reducing the number of ova shed. These results indicate that the NOS activity and NOS positive cells increased after hCG injection, and that eNOS was likely the main NOS increasing in the ovary during ovulation. It is concluded that NO produced between 5 and 9h after hCG might play a supportive role in ovulation.

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Multiple endocrine neoplasia type 1(MEN1) is a human hereditary tumor syndrome characterized by the development of endocrine adenomas of the parathyroid, anterior pituitary, and enteropancreatic tissue. Several lines of evidence have implicated the recently identified MEN1 gene located on chromosome 11q13 as a recessive tumor suppressor gene. Here, we analyzed MEN1 wild-type gene expression in tumors from a large MEN1 kindred. A deletion of codons 227-228 (678de16) located in exon 4 was found in tumor and peripheral blood complementary DNA using a simplified single-strand conformational polymorphism (SSCP) approach well suited for clinical MEN1 mutation screening. The identified 678de16 cDNA mutation deletes a potential phosphorylation site (Tyr227) and corresponds to a germ line mutation co-segregating with disease phenotype in this MEN1 family. Loss of heterozygosity analysis by fluorescent microsatellite PCR showed an exclusive loss of the MEN1 wild-type (and retention of the mutated) allele detectable in DNA from microdissected parathyroid and pancreatic, but not in adrenal, adenomas. Our findings confirm the synergism between MEN1 gene mutations and subsequent MEN1 allelic losses in the tumorigenesis of MEN1-associated adenomas.

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Transforming growth factor-β1 (TGF-β1) stimulates articular chondrocyte cell proliferation and extracellular matrix formation. We reported previously that immediate and transient expression of c-fos mRNA through protein kinase C activation is required for the mitogenic effect of TGF-β1 on cultured rat articular chondrocytes (CRAC). In gel kinase assays using myelin basic protein (MBP) showed that total cell lysates from cells treated with TGF-β1 caused rapid phosphorylation of MBP, which suggests the involvement of mitogen-activated protein kinase (MAPK) activation. To identify specific MAPK pathways activated by TGF-β1, we performed in vitro kinase assays using specific substrates. TGF-β1 induced a rapid activation of extracellular signal regulated kinase (ERK) with a peak at 5min, which decreased to basal levels within 240min after TGF-β1 stimulation. In contrast, the c-jun N-terminal kinase activity increased only about 2.5-fold after 240min of stimulation and p38 MAPK activity did not change significantly. ERK activation by TGF-β1 was also confirmed by in vivo phosphorylation assays of Elk1. However, a specific MEK1 inhibitor, PD98059, significantly decreased TGF-β1 induced Elk1 phosphorylation in a dose-dependent manner. Furthermore, PD98059 reduced the TGF-β1-induced cell growth by 40%. These results indicate that TGF-β1 specifically activates MEK1 and subsequent ERK pathways in CRAC, and that the activation of this MAPK pathway plays a role in the mitogenic response to TGF-β1.

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To clarify the effect of GH on the development of seminiferous tubules in premature male rats, we investigated whether GH accelerates spermatogenesis under the condition of gonadotropin deprivation. Male Wistar rats aged three weeks were divided into three groups and subjected to administration of either long-acting GnRH agonist (GnRHa) or a combination of GnRHa and rat GH, with normal saline solution as control. After the 4-week treatment, sperm density and motility in the right epididymis were measured and seminiferous tubules of right testes were histologically examined. Sperm density and motility were significantly higher in GnRHa+GH-treated rats than in GnRHa-treated rats. In histological examination, the numbers of germ cells in various stages were increased in GnRHa+GH-treated rats compared with GnRHa-treated rats, with the number of mature spermatid being noticeably higher in GnRHa+GH-treated rats. These results suggest that administration of GH decreases loss of germ cells at various stages of spermatogenesis under the condition of gonadotropin withdrawal.

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A 57-yr-old female with corticotropinoma showing no Cushingoid stigmata is reported. Basal plasma levels of ACTH measured with immunoradiometric assay and β-endorphin were high, 12.6-15.9pmol/l and 3.5 pmol/l, respectively. Plasma cortisol level and urinary free cortisol excretion were normal, 303-359nmol/l and 171-226nmol/day, respectively. Plasma ACTH markedly increased to 70.5pmol/l with intravenous administration of 100μg CRH. Diurnal rhythm of plasma ACTH was seen, but its level in the night was still high. Plasma ACTH suppression with dexamethasone was insufficient. CRH stimulation after dexamethasone suppression increased plasma ACTH level from 4.4 to 13.7pmol/l. Intravenous administration of 4μg desmopressin increased plasma ACTH from 15.6 to 19.6pmol/l. Oral administration of 16mg lepramide insufficiently decreased plasma ACTH from 7.3 to 5.3pmol/l. However, plasma cortisol responses in these conditions were normal. Postoperative pathological study revealed subtype 1 corticotropinoma immunohistochemically and electron-microscopically. Postoperative basal plasma ACTH decreased to 3.9pmol/l, although plasma cortisol did not change. Diurnal rhythm and dexamethasone suppressibility of plasma ACTH became normal. Plasma sample was chromatographed on a Sephadex G-75 column. The elution profile showed two peaks of ACTH, one of which was compatible with 1-39 ACTH and another with higher molecular weight ACTH which was probably secreted from corticotropinoma. Anomaly in processing of proopiomelanocortin was suspected.

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In this study, nine patients with Graves' ophthalmopathy with positive clinical activity score (CAS), who were either unresponsive or not suitable for glucocorticoid treatment, were given 100μg of octreotide three times daily, subcutaneously, for three months. The mean age was 49±13 years. All patients were under either propylthiouracil or methimazole therapy and were euthyroid for at least one month prior to the start of the octreotide treatment. The mean degree of proptosis as measured with the Hertel exophthalmometer decreased slightly after the treatment (22.0±3.0 vs 19.6±2.4 for the right eye and 22.2±1.9 vs 20.2±2.2 for the left eye; p<0.05). The mean activity score decreased from 3.2±0.8 to 1.7±1.1 (p< 0.005) and the mean score of eye signs according to the NOSPECS classification showed improvement with octreotide therapy (3.2±0.7 vs. 2.2±1.4; p<0.05). Seven patients responded favorably to octreotide treatment. In the remaining two no improvement was observed. Four of the responders could be followed up for 20 months after the treatment and all maintained the favorable state of eye findings obtained with octreotide. We conclude that octreotide seems to be a safe and effective drug in Graves' ophthalmopathy, especially in improving soft tissue involvement, and can be used in patients who are unresponsive to glucocorticoid treatment or who cannot use these drugs for some reason.

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Here we report a case of levothyroxine-induced liver dysfunction. T₄ (levothyroxine) has been more commonly used for the treatment of hypothyroidism than T₃ active hormone (triiodothyronine), because with the former drug a stabler plasma concentration is obtained after oral administration. Although there are few reports on levothyroxine-induced liver dysfunction, we treated a primary hypothyroid patient with high serum aminotransferase after administration of levothyroxine. Liver dysfunction was improved after cessation of the drug administration. Antibody to T₄ was found in the serum of the patient after this event. From clinical course and laboratory data of the patient, the episode of liver damage was considered to be induced by levothyroxine. We then administrated triiodothyronine, and it did not induce liver dysfunction. Changing levothyroxine to triiodothyronine resulted in a successful clinical course in this case, as re-administration of the doubtful drug is strictly limited.

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Sleep-disordered breathing (SDB) is common in patients with growth hormone (GH) secreting pituitary adenomas. Since long-term untreated SDB aggravates systemic conditions (hypertension and arrhythmia etc.), the therapeutic outcome of SDB is important in reducing morbidity and mortality rates. But the results of a quantitative analysis of the lowered GH and IGF-1 levels in SDB in a relatively large number of patients are not detailed. Ten consecutive acromegalic patients were studied with a bedside oximeter. Preoperatively they were divided into two groups based on the presence (SDB group=6 patients) or absence (non-SDB group=4 patients) of clinical symptoms of SDB such as habitual snoring, excessive daytime somnolence and nocturnal apneic episodes. The serum IGF-1 averaged 931.7ng/ml in SDB group and 898.3ng/ml in non-SDB group. The oxygen desaturation index (ODI) (the number of oxygen desaturations exceeding 4% from the base line) was 29.1+/-15.4 in the SDB group and 2.5+/-1.8 in the non- SDB group (P=0.01). Other oximeter parameters such as the percent of the time spent at O₂ saturation <90% and the mean and the lowest O₂ saturations closely correlated with the degree of the clinical symptoms. A postoperative sleep study was conducted in 5 patients in the preoperative SDB group, 4 months or more after the surgery. The serum GH and IGF-1 levels normalized in 3 patients but remained slightly high in 2. ODI became 9.1+/-5.6, which was significantly lower than the preoperative value (P=0.026). One patient had a complete clinical resolution. The other 4 obtained slight to moderate improvement clinically and oximetrically despite normalized or decreased hormonal levels. This study clarified that the response of SDB to lowering of the GH level varies from one patient to another and persisting SDB despite the normalization of the hormonal levels suggests the involvement of other factors in the production of SDB.

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A 73-year-old man with general malaise and nausea following a common cold diagnosed by a local physician was found to have multiple hepatocellular carcinomas with enlarged bilateral adrenal glands, combined with adrenal insufficiency. Hydrocortisone replacement improved the symptoms and laboratory findings. Autopsy findings revealed that each adrenal gland was completely replaced by the tumor measuring 11cm in diameter, and no adrenal tissue was recognized. Histologically, the adrenal tumors, as well as the liver tumors, were moderately differentiated Edmondson type II hepatocellular carcinomas. This is a second report of adrenal insufficiency due to hepatocellular carcinoma as a primary site of metastatic adrenal tumor.

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This study investigated the effects of reconstituted basement membrane Matrigel on the proliferation and prolactin expression of GH₃ cells in culture for 6 days. When cells were cultured on Matrigel, the initial attachment was increased but the cell number was not changed with time whereas rapid increase in cell number was observed in cultures on plastic. Bromodeoxyuridine (BrdU)-labeling showed that BrdU incorporation ratio of GH₃ cells cultured on Matrigel was about one half of that observed with cells cultured on plastic (9.7±0.7% vs. 18.7±1.2%). Immunocytochemistry revealed that the ratio of the prolactin-immunoreactive GH₃ cells was about 3.6 times (58.4± 2.9% on Matrigel vs. 16.2±1.4% on plastic), which was compatible with the results of Western blot analysis. In situ hybridization demonstrated that prolactin mRNA-positive cells were identified more frequently when cells were cultured on Matrigel compared to cultures on plastic. These findings indicate that Matrigel is a proper culture substrate for the long-term culture of GH₃ pituitary cells due to the inhibition of overgrowth and promotion of prolactin expression.

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To evaluate pharmacokinetics of growth hormone (GH) and its effects on IGF-I, glucose, insulin, nonesterified fatty acid (NEFA) and riglyceride (TG), fifteen Japanese healthy adult male volunteers (20-27 years old) were studied. The subjects were divided into three groups, and received with a single s.c. injection of 0.075, 0.15 and 0.30IU/kg of GH, respectively. The subjects assigned to receive 0.30IU/kg were administered for additional 6 days. After a single administration of GH, Cmax and AUC of GH were increased in a dose-dependent manner. There was a significant positive correlation between the AUC and the T1/2 (r=0.516, P<0.05). Total body clearance was significantly greater in 0.075IU/kg group than the other groups and showed a significant negative correlation with Cmax (r=-0.694, P<0.005) and AUC (r=-0.723, P<0.005). After a single administration of each dose, serum IGF-I concentrations were increased gradually. In the repeated administered group (0.30IU/kg), IGF-I concentrations almost reached a plateau at a significantly high level four days after the start of administration and remained at a high level (786-405.4ng/ml) until day 8. There was no significant difference in diurnal change of blood glucose and serum insulin after a single administration of GH among three groups. In the 0.3U/kg group, there was no significant difference in diurnal change of blood glucose between day 1 and day 7, but serum insulin level was significantly higher in day 7 than in day 1 (P<0.01). Serum concentrations of NEFA were increased over time after administration in all subjects administered once or repeatedly. TG concentrations showed no changes after single administration of each dose level, but were significantly increased on day 7 in the subjects repeatedly treated with 0.30 IU/kg/day. This effect is speculated to be caused by high dose GH treatment. The above findings demonstrated that higher GH dose significantly influences on carbohydrate and lipid metablism. It remains necessary to elucidate what kinds of effects of the long-lasting increased levels of insulin and triglyceride, even if reversible, would have on glucose and lipid metabolism.

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T₄-binding globulin (TBG) is the major thyroid hormone transport protein in humans. Inherited abnormalities in the level of serum TBG have been classified as partial deficiency, complete deficiency and excess. A single nucleotide deletion or substitution in the TBG gene, located on Xq22, has been detected in partial and complete deficiencies. As for inherited TBG excess, the gene amplification has been recognized in two Japanese families recently. In this study, an additional three Japanese families, one familial (F-I) and two sporadic TBG excess (F-II, F-III), were analyzed. Serum TBG levels in hemizygous males were 73, 47 and 42μg/ml, three- to two-fold the normal value. The molecule had normal properties in terms of heat stability and isoelectric focussing pattern. The gene dosage of TBG was evaluated by coamplification with autosomal βGlobin or X-chromosomal Duchenne Muscular Dystrophy (DMD) and subsequent quantitation by HPLC. The TBG/βGlobin ratios of the affected male and female of F-I were 3.09- and 3.86-times, respectively, compared to that of the normal males. The TBG/DMD ratios were 2.93- and 2.09-times, respectively. These results are compatible with three copies of the TBG gene on the affected X-chromosome. Similarly, a twofold increase in gene dosage was demonstrated in the affected males of sporadic cases. Their mothers with normal TBG values had the same TBG gene dosage as normal females, suggesting that de novo gene duplication arose in gametes probably during meiosis. Amplification of the TBG gene was not recognized in these three families by in situ hybridization of prometaphase chromosomes. Though the mechanism remains unproved, gene amplification of TBG was considered to be a common cause for inherited TBG excess.

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