The effects of GH injections on the concentrations of plasma hormones and metabolic substrates in pigs were studied under various nutritional conditions. In experiment 1, four pigs (Large white, barrows, 108 days old) were maintained at three feeding levels: (1) high level (8, 501±46kcal/day), (2) maintenance level (2, 104±44kcal/day), and (3) fasting (last meal was on day -2). The bovine GH (100μg/kg BW) was subcutaneously injected on experiment days 0, 1, and 2. In experiment 2, four pigs (Landrase, barrows, 120 days old) were maintained at the high feeding level and the bovine GH (100μg/kg BW) was injected 2, 26, and 50h after the last meal. The plasma hormone and metabolic substrate concentrations were measured. In experiment 1, the plasma IGF-I concentrations increased on days 1-4 in the high feeding level and maintenance fed pigs, but did not increase in the fasting pigs. The plasma glucose concentrations increased after the GH injection in the high feeding level and maintenance fed animals. The plasma NEFA concentrations increased after the GH injection in the maintenance fed and fasting animals. In experiment 2, the plasma IGF-I gradually decreased after the last meal. The GH injection administered 26 or 50h after the last meal still produced an increase in the plasma IGF-I levels. These data clearly show that the effect of GH was modified depending on the nutritional condition of the pigs.
2-Hydroxylation is one of the major metabolic pathways of estrogens and is believed to be catalyzed by a form of cytochrome P450. Recently it has been reported that estrogen 2-hydroxylase activity in human placenta is catalyzed by aromatase. Some investigators suggested the effect of catechol estrogen on human placental steroidogenesis which may be related to pregnancy-induced hypertension (PIH) through the inhibition of catechol-O-methyltransferase (COMT) activity. In order to better understand the interrelationship between placental aromatase and estrogen 2-hydroxylase activities in PIH patients, both activities were evaluated in the PIH placentas. Human placental microsomes obtained from PIH patients were incubated with [1β-3H]androstenedione or [2-3H]estradiol in the presence of NADPH. Aromatase and estrogen 2-hydroxylase activities were assessed by the tritium water method. The immunosuppression patterns of both activities due to monoclonal anti-aromatase cytochrome P450 antibody (MAb3-2C2) were studied. Estrogen 2-hydroxylase activity was significantly higher in PIH placentas (4.7± 0.9pmol/min/mg protein, n=7) than in normal placentas (3.0 ±0.7pmol/min/mg protein, n=7). When the PIH placental microsomes were subjected to immunosuppression by 1 to 100μg IgG of MAb3-2C2, estrogen 2-hydroxylase activity was suppressed by 94 to 65% whereas aromatase activity was strongly suppressed by 72 to 17%, respectively. From our results of high estrogen 2-hydroxylase activity in PIH placentas, it is assumed that there is a different estrogen catalyzing mechanism in PIH placentas.
The role of mesencephalic raphe nuclei in the induction of pseudopregnancy was investigated in female rats. The dorsal or median raphe nucleus lesions (DRL or MRL, respectively) were made by means of a radiofrequency lesion generator. Two or 3 weeks after the operation, in order to induce pseudopregnancy, the vagina was stimulated electrically on the day of proestrus or 1mg/kg b.w. reserpine was injected on the day of diestrus I. Traumatization by passing thread to one uterine horn was performed to induce deciduoma 5 days after vaginal stimulation or 3 days after reserpine injection. As the results, decidual response was seen in most control and sham females in both vaginal stimulation and reserpine-treated groups. In contrast, incidences of deciduoma in DRL females with vaginal stimulation or reserpine-injection were significantly lower than those in control and sham groups. In the MRL females with either vaginal stimulation or reserpine-treatment, incidences of deciduoma were comparable to those of the control and sham operated groups. These results suggest that the dorsal raphe nucleus plays an important role in pseudopregnancy-inducing mechanisms in female rats.
We immunohistochemically investigated the localization of activin A and follistatin in various human tissues with specific antibodies to recombinant human (rh-) activin A and rh-follistatin. Specific immunostaining of activin A was detected in Leydig and Sertoli cells of the testis. In the ovary, granulosa cells of mature follicle and luteal cells of the corpus luteum stained for activin A. Immunoreactive activin A was present in somatotrophs of the pituitary gland and insulin-positive B cells of the pancreatic islets. Immunoreactivity for activin A was also found in thyroid follicular cells, adrenocortical cells, neuronal cells of the cerebrum and monocytoid cells in the bone marrow. Follistatin, an activin-binding protein, was immunostained in the same tissues as activin A. These findings indicated that activin A and follistatin are widely distributed in human tissues, suggesting that activin plays important roles as a common regulator in various tissues under the control of co-existing follistatin.
The reported number of adrenal incidentalomas has been increasing because of wider application of imaging techniques. Patients with asymptomatic cortisol producing adrenal adenoma (ASCA) which secretes cortisol without clinical evidence of Cushing's syndrome has been more frequently observed than previously assumed, and they have a risk of adrenal insufficiency after adrenalectomy. Therefore patients with incidentalomas should be screened for cortisol overproduction. The aim of this study is to discover an easy screening test to uncover ASCA. We investigated the hormone profiles of 4 patients with ASCA in comparison with 11 patients with non-functional adrenal tumor and 10 patients with adrenal Cushing's syndrome. We also investigated the expression of dehydroepiandrosterone sulfotransferase (DHEA-ST) in surgically removed attached non-neoplastic adrenal tissues by immunostaining, which was considered to represent the degree of suppression of the hypothalamo-pituitary-adrenal axis. Serum dehydroepiandrosterone sulfate (DHEA-S) levels of all the patients with ASCA and adrenal Cushing's syndrome were lower than those of healthy subjects of corresponding age, but they were within the normal range in the patients with non-functional adrenal tumors. The serum DHEA-S level reflects the degree of suppression of the normal adrenal gland by cortisol hypersecretion from adrenal tumors. But the serum level of DHEA-S decreases with age, and because the normal range of serum DHEA-S is low in elderly subjects, we should be careful to evaluate the level of DHEA-S in elderly patients with adrenal Cushing's syndrome or ASCA. The immunohistochemical study showed DHEA-ST expression was noticeably suppressed in the adjacent adrenal cortex in ASCA and adrenal Cushing's syndrome. The decreased expression of DHEA-ST may reflect autonomous neoplastic cortisol secretion and subsequent ACTH suppression in ASCA and adrenal Cushing's syndrome. A single measurement of plasma ACTH or measurement of ACTH response to corticotropin-releasing hormone was not enough to screen for ASCA because of the wide variation among the cases. Dexamethasone suppression test is essential in identifying ASCA and also a single determination of serum DHEA-S is easy and may be useful for the screening of ASCA in adrenal incidentalomas in young and middle aged subjects, and is especially useful for outpatients.
Changes in magnesium metabolism, along with those in sodium, were investigated in 17 patients with Graves' disease (14 females and 3 males, mean ± SD, 44.8±12.2 years) and their relationship to plasma levels of thyroid hormones were assessed before and after treatment. Each patient was studied in hyperthyroid state and euthyroid state after treatment with methimazole. Treatment with methimazole increased the magnesium concentration both in erythrocytes (2.00±0.18 vs. 2.08±0.24mmol/l cells, P<0.05) and in serum (0.72±0.12 vs. 0.84±0.11mmol/l, P<0.001) but both urinary output and fractional excretion of magnesium decreased significantly (P<0.05 and P<0.001, respectively). The erythrocyte sodium concentration decreased with treatment (10.7±2.6 vs. 8.1±1.1 mmol/l cells, P<0.001) but the serum sodium remained unchanged (140.9±1.9 vs. 140.9±2.1mmol/l, NS). Urinary excretion of sodium also decreased with treatment (P<0.05), but only changes in indices of magnesium metabolism (decrease in renal fractional excretion, rise in serum level) correlated significantly with those of the thyroid functions with treatment. These observations clearly indicate that in Graves' disease, the magnitude of magnesium metabolism alteration is closely related to the extent of the increase in thyroid hormones in plasma.
Recent studies have demonstrated that inhibins and activins both play not only endocrine roles but also local regulatory roles in gonadotoropin secretion. There has been controversy as to the subtype of rat pituitary inhibin/activin. We studied the levels of inhibin α, βA and βB subunit mRNAs by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) and the changes in their levels by adding inhibin α, βA and βB mRNA antisense oligonucleotides and inhibin A, activin A or GnRH to cultured rat anterior pituitary cells. This study demonstrated the level of 3 mRNAs to be 1.6× 10-2, 0.75 and 3.4×10-2 molecules/cell with a molar ratio of 1:50:2. A stimulatory role for activin B in FSH secretion was suggested as βB mRNA antisense oligonucleotide decreased FSH secretion. The βB mRNA level tended to be decreased by the addition of activin A, but the decrease was not statistically significant. GnRH did not affect α and βB mRNA levels when administered singly. The level of βA mRNA was not changed by any of the above treatments. In conclusion, the presence of inhibin α, βA and βB subunit mRNAs in the rat anterior pituitary with the greatest abundance of βA was demonstrated by using RT-PCR. Activin B or activin AB may play important roles in FSH secretion in an autocrine or a paracrine fashion, and activin A may play an indirect role in FSH secretion.
In order to clarify whether the long-term effect of estrogen on bone mineral density (BMD) is reinforced by low dose calcium supplements, 600-800mg of calcium lactate was administered to postmenopausal or oophorectomized women who had been undergoing unopposed estrogen therapy for at least 2 years and whose serum calcium level was suppressed to below the normal range. To patients whose serum calcium levels had been within the normal range, the same dose of estrogen alone was continued. Changes in lumbar spine BMD before and after calcium supplementation was measured by dual-energy X-ray absorptiometry. Lumbar spine BMD decreased by -0.37% for 2 years in women treated with estrogen alone, while that of women treated with estrogen and calcium increased by 2.78% (P=0.003). These results indicate that low dose calcium supplements potentiate the effect of estrogen in women with decreased serum calcium during long-term hormone replacement therapy.
We here describe two patients with empty sella syndrome who had variable and abnormal ACTH and cortisol secretory profiles. The patients are a 58-year old male and a 41-year old female, both of whom were neurotic. In both cases, low-dose dexamethasone suppression tests (1mg, p.o., overnight) caused variable responses, such as a paradoxical increase, insufficient decrease or normal decrease in ACTH and cortisol depending on the period when they were performed. The circadian rhythm of ACTH and Cortisol also showed variable patterns. Abdominal CT showed slight enlargement of both adrenal glands. Adrenal scintigraphy after dexamethasone suppression (3mg, p.o., 7 days) revealed uptake into both adrenal glands. MRI of the brain indicated empty sella, but failed to show evidence of pituitary adenoma. It remains to be elucidated whether these secretory profiles of ACTH and cortisol in the two cases are only to be regarded as secondary changes associated with neurosis, as reported in depression or alcoholism, or the two cases share some pathogenetic mechanism with cyclic Cushing disease.
To analyze how the synthesis and release of glycosylated PRL (G-PRL) is regulated, we transfected human PRL complementary deoxyribonucleic acid (cDNA) into three different cell lines consisting of GH3 cells that originated in rat pituitary tissue, Chinese hamster ovary cells, and COS-1 cells generated from monkey renal tissue. 35S-labeled PRLs produced by the cells were immunoprecipitated with anti-human PRL antiserum, and the ratios of G-PRL to total PRL were compared. PRLs of 23kDa and 25kDa were detected in the cell lysate and medium. The 25-kDa PRL was confirmed to be a glycosylated form by endoglycosidase treatments. The ratios of G-PRL/total PRL were 0.17-0.33, which were similar in lysates and media and among different cell lines. Pulse-chasee xperiments revealed that the autonomaous secretion rates of G-PRL and non-glycosylated PRL were almost identical. These results indicate that synthesis and secretion kinetics of human PRL may not be affected by its glycosylation in the cells transfected with PRL cDNA.
Inhibins and activins have been known to modify the secretion of various pituitary hormones. To study whether inhibins and activins are present in human pituitary tissues, immunohistochemical studies with antisera to activin A and inhibin a subunit were performed on 9 human pituitary adenoma tissue specimens and one sample of normal pituitary tissue adjacent to one adenoma. Activin immunoreactivities were demonstrated in the cytoplasms of one GH and one PRL and two non-functioning adenomas and one normal pituitary tissue, but they were negative in one PRL, one ACTH, one FSH and two non-functioning adenomas. Thus, the presence and absence of activin in the same type of adenoma in regard to hormone production, suggested that the difference in immunostaining simply reflected the difference in the activin concentration. In contrast to this, inhibin α subunit immunoreactivity was not found in any of the tissues studied. These data suggested a local synthesis of activin in the normal pituitary as well as various kinds of pituitary adenoma tissues and its local role in the human pituitary gland.
In two patients with lymphocytic adenohypophysitis, images of the pituitary gland were serially observed by MRI. In both cases, the pituitary gland had swollen during the late stage of the first pregnancy. In case 1, MRI findings were representative of lymphocytic adenohypophysitis. After delivery, plasma levels of PRL, ACTH and cortisol decreased markedly. The height of the pituitary gland gradually decreased from 22mm (14 days after delivery) to 13mm (73 days) and became rapidly smaller (4.9mm, 115 days) following administration of massive doses of hydrocortisone for the treatment of acute adrenal insufficiency induced by painless thyroiditis. Six years later, the height was 2.5mm. Low plasma levels of PRL and cortisol persisted. Diabetes insipidus did not develop. In case 2, MRI revealed a pituitary mass accompanied by a cystic change. Lymphocytic adenohypophysitis was confirmed by histological examination. Because pituitary function tests indicated that ACTH, PRL, GH and TSH were of low levels, hydrocortisone and L-thyroxine were orally administered. No diabetes insipidus was demonstrated. MRI disclosed that the height of the pituitary gland was 23mm (17 days after delivery) but decreased to 17 and 5.5mm after 44 and 128 days, respectively. Four years later immediately after the second delivery, it was 1mm, and the patient was diagnosed as having empty sella. Long-term observation of lymphocytic adenohypophysitis demonstrated that the pituitary gland was markedly atrophied, leading to empty sella. It is believed that some of the classic cases of Sheehan's syndrome associated with empty sella may include lymphocytic adenohypophysitis.
To investigate the possible humoral factor(s) influencing thyroid cell activity in chronic renal failure, we measured serum activity which stimulates or inhibits the [3H]thymidine incorporation by using a cultured functioning rat thyroid cell line (FRTL-5 cells) in 17 patients on hemodialysis and 19 healthy controls. Polyethylene glycol-treated serum was centrifuged and FRTL-5 cells were cultured with the supernatant. Thyroid stimulating activity was determined by [3H]thymidine incorporation after incubation for 72h. There was no significant difference in [3H]thymidine incorporation between cultures incubated with patient and normal serum, suggesting the absence of the stimulating activity. But when patient serum was added to cultures together with 20 or 50μU/ml of TSH, the TSH-stimulated increase in [3H]thymidine incorporation was significantly decreased, indicating the presence of thyroid inhibiting activity, which possibly inhibits the thyroid cell growth. This activity was not significantly altered by hemodialysis. No significant correlation was observed between this activity and serum levels of thyroid hormones or the iodine concentration. Patients on hemodialysis therefore have serum thyroid inhibiting activity which is nondialysable, differs from iodine, and could influence the thyroid cell growth.
To study GH response to the long-acting somatostatin analogue, we treated 11 actively acromegalic patients with octreotide (Sandostatin), 100μg, sc, tid, for six months. Their endocrinological outcomes and clinical improvements varied. The 11-h GH secretory profiles on pretreatment day confirmed the hypersecretion of GH in all patients. Three hours after the first dose of octreotide, serum GH declined rapidly to levels below 5ng/ml in all but two patients who failed to normalize their serum GH. In spite of the subsequent doses, there was no further suppression in serum GH. Drug resistance with GH rebound developed in some patients after three months of continued treatment. The paradoxical serum GH rises in response to oral glucose or iv TRH detected before the treatment in all patients attenuated or disappeared after the 6-month octreotide therapy; an exceptional case was one of the above-mentioned two patients, whose serum GH was stimulated more than before by glucose and TRH at the end of therapy. Serum insulin-like growth factor I (IGF-I) levels of all patients showed a significant reduction after 6-month treatment, but their mean values remained abnormally high. There were no intolerable adverse side effects; some patients, however, experienced pain at the injection site, passage of loose stool, and incidence of new gall stone or intrahepatic lesions on octreotide therapy. We concluded that octreotide was a useful long-term adjunctive therapeutic agent for patients with active acromegaly, but that a high degree of response heterogeneity including total refractoriness would be expected.