Insulin receptors on hepatocytes were studied in spontaneously diabetic Chinese hamsters, which are the animal models for insulin deficient diabetes. Insulin binding in diabetic animals increased mainly due to an increase in the number of receptors. Although binding affinity of diabetic animals was similar that of control animals, a kinetic study revealed that both the association constant and the dissociation rate constant decreased in diabetic animals. Negatively cooperative interactions between receptors were demonstrated in control and diabetic animals, and both the magnitude and sensitivity of this effect was the same in both types of animals. A significant inverse correlation between insulin binding and the plasma insulin concentration was found in these animals. These results therefore suggest that there is an increase in the insulin binding in the insulin deficient diabetic state mainly due to an increase the number of receptors with a decrease in both the association and dissociation rate constants, and these changes may be important in the altered metabolic state.

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Serum concentrations of 17-hydroxypregnenolone, 17-hydroxypregnenolone sulfate and 17-hydroxyprogesterone were measured simultaneously in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, using a combined radioimmunoassay method. All these precursor steroids were found to be markedly elevated in the sera of untreated patients with a salt-losing form of the disease, whereas, in untreated patients with a simple virilizing form, only the concentration of unconjugated steroids was increased and the 17-hydroxypregnenolone sulfate concentration remained within the normal range. Among the patients with a salt-losing form under maintenance therapy, these steroids were all still significantly increased in those on insufficient control, whereas only 17-hydroxyprogesterone was significantly but slightly increased in those on adequate control. Although the mechanism whereby the serum 17-hydroxypregnenolone sulfate concentration is not increased in the untreated simple virilizers is unknown, both a milder degree of 21-hydroxylase deficiency and a role of 17-hydroxypregnenolone sulfate in adrenal steroid production as a kind of supplier are suggested as possible explanations, especially in the neonatal period and early infancy. Thus, this study showed the serum concentrations of 17-hydroxypregnenolone and its sulfate together with 17-hydroxyprogesterone in patients with 21-hydroxylase deficiency in various conditions.

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Epidermal growth factor (EGF), a potent mitogenic peptide, is known to be present in the fluid of the uterine cavity. Recent studies have demonstrated the messenger RNA for EGF in the rat uterus. Therefore, in an attempt to clarify its physiological role, we investigated the receptors for EGF in human endometrial tissues. The particulate fractions from endometrium possessed the capacity to bind EGF in a specific, saturable and reversible manner. The Scatchard plot was linear, showing a single class of the receptor with an apparent Kd of 3.8×10^-9 M. The amount of specific EGF binding was very low during menstruation and increased gradually, reaching its peak in the late follicular phase. There was an abrupt decline in the binding after ovulation with no change in the Kd value. These results imply the possible involvement of EGF in the process of proliferation of human endometrial tissues.

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Ten women of polycystic ovarian-type (PCO-type) anovulation, having a decreased ratio of FSH to LH and androgen excess, resistant to the previous clomiphene, bromocriptine and/or daily im injections of human menopausal gonadotropine (hMG), were treated with continuous sc infusion of 150 IU/day hMG. The treatment was initiated on cycle day 2-5 and continued until the dominant follicle reached 20mm or more in diameter, when an im bolus of 10, 000 IU human chorionic gonadotropin was given. The treatment elevated the geometric mean of pretreatment serum FSH (8.6mIU/ml) to 15.9mIU/ml (p<0.001), while serum LH decreased from 29.4mIU/ ml to 20.7mIU/ml (p<0.01). This resulted in a highly significant increase in the FSH/LH ratio from 0.29 to 0.77 (p<0.0001). Follicle enlargement was demonstrated in 13 of the 14 treatment cycles, 12 of which were ovulatory. Pregnancy ensued in 4 cases, 1 of which was a quadruplet pregnancy. Continuous infusion of hMG was indicated as an effective way of inducing ovulation in PCO-type anovulation resistant to conventional methods of ovulation induction.

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The serum bone Gla protein (BGP) level was measured in patients with idiopathic hypoparathyroidism, and primary hyperparathyroidism, and normal volunteers. The mean serum BGP level was 4.5±0.20μg/l in 40 normal volunteers. It was significantly lower in 12 patients with idiopathic hypoparathyroidism (1.6±0.21μg/l, p<0.001) and significantly higher in 33 patients with primary hyperparathyroidism (13.0±1.3μg/1, p<0.001).
When a single intravenous injection of 30μg of human PTH 1-34 was administered to the patients with idiopathic hypoparathyroidism, there was no significant change in serum BGP within the next 24 hours. Following a therapeutic oral dose of alfacalcidol, serum BGP was appreciably increased (p<0.001) from the preadministration value of 1.6±0.21μg/l to 3.9±0.34μg/l.
In patients with primary hyperparathyroidism, the surgical excision of parathyroid adenoma led to a sharp decrease in serum PTH but a gradual decrease in serum BGP. The latter approximately paralleled the decline in serum alkaline phosphatase.
Thus, serum BGP is a marker that reflects bone turnover status in parathyroid disease. It appears that the active form of vitamin D directly increases the secretion of BGP in existing osteoblasts and PTH mainly affects serum BGP to stimulate the bone remodeling cycles with its long term effect.

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Twenty-six anovulatory women of polycystic ovarian (PCO)-type were treated with bromocriptine (Br) at a daily dose of 5mg for 2months. Ovulatory cycles were resumed in 18 (69.2%) women (Br-responders). No difference between pretreatment serum levels of FSH, LH, PRL and estradiol and FSH/LH ratios in Br-responders and nonresponders was observed. The geometric mean of circulating androstenedione (A-dione) in Br-responders (2.58ng/ml) appeared higher than that in nonresponders (2.11ng/ml) but was not statistically significant. The geometric mean of dehydroepiandtrosterone sulfate (DHEA-S) in Br-responders (1652ng/ml) was lower (p<0.01) than that in nonresponders (2582ng/ml). The ratio of DHEA-S to A-dione (D/A ratio) exhibited a highly significant between-group difference (p<0.001)(646 and 1222 for Br-responders and nonresponders, respectively). Br-nonresponders with high DHEA-S levels and D/A ratios tended to hyperproduction of adrenalandrogen, and Brresponders with high A-dione levels and low D/A ratios to hyperproduction of ovarian androgen. The present study indicates that Br is effective in PCOtype women presumably with ovarian androgen hyperproduction. The efficacy of Br, when applied to PCO-type women, could be predicted with their D/A ratios.

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Changes in uterine weight and the estrogen receptor concentrations were examined in persistent estrous (PE) and persistent diestrous (PD) rats at 80 days of age. To prepare PE rats, 100μg estradiol benzoate (EB) was injected sc into 3-day-old females. PD rats were obtained by daily injections of 10μg EB into females for 10 consecutive days from the day of birth. The uterine weight in PE rats at 80 days was comparable to that in metestrous controls. The uteri of PD rats were smaller than those in PE rats. The concentrations of estrogen receptor in nuclear fractions in PE and PD rats were much lower than those in proestrous controls. Receptor concentrations in cytosol fractions were significantly lower in PE and PD rats than in control diestrous, proestrous and estrous rats. The dissociation constants and sedimentation coefficients of estrogen receptors in PE and PD rats were found to be in the same range as those in control rats. Thus, the reduction in the activity of cytosol receptors in these rats is attributable to a quantitative change in the amount of estrogen receptor protein. To study the response of the uterus to estrogen, ovariectomized rats were injected daily with 10 pg estradiol for 7 consecutive days. The uterine growth of PE and PD rats after administration of estradiol was less marked than in controls, indicating a reduction of estrogen sensitivity of the uterus. Seven daily administrations of estradiol continued to increase the concentration of uterine cytosol estrogen receptor in controls. In contrast, in PE and PD rats, the receptor concentrations continued to increase during the first 3days.and then remained constant.These data suggest that EB in neonatal treatment may directly affect the mechanism of receptor synthesis in uterine tissues. This effect may contribute to the reduction of the uterine response to estrogen.

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We studied the plasma GH profiles in 6 patients with Turner's syndrome and 6 normal girls of short stature by sampling every 20 min for 24 hours. We observed episodic secretion of GH in these subjects. The mean plasma 24 h GH level in patients with Turner's syndrome was 3.6±1.4 (SD) ng/ml which was significantly lower than that of normal short girls (7.1±2.2ng/ml, p<0.01). The GH secretion during both nighttime and daytime was decreased in the patients with Turner's syndrome, however the number of pulses did not differ significantly. There were no correlations between the mean plasma 24h GH level on one hand and peak GH level obtained after GH provocative test and plasma somatomedin C on the other.
Plasma FSH and LH levels were also measured in 4 patients with Turner's syndrome. Both levels were elevated and there observed no clear pulsatilesecretion of FSH, but, some pulsatile secretion of LH was observed in two patients.
These data indicate that patients with Turner's syndrome have decreased endogenous GH secretion, even though they show normal GH responses to GH provocative tests.

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Thermogenesis of brown adipose tissue (BAT) of genetically obese mice, KKA^Y mice, was examined by measuring the BAT mitochondrial guanosine diphosphate (GDP) binding as an index of thermogenesis and comparing it with that of normal C57BL mice. No great difference in GDP binding was observed in KKA^Y and C57BL mice fed a stock diet. However, when they were given a sucrose solution, the increase in BAT mitochondrial GDP binding of KKA^Y mice (+22%) was much lower than that of C57BL mice (+106%). A high fat diet increased BAT mitochondrial GDP binding in KKA^Y mice to the same extent (+82%) as in C57BL mice. When the mice were fasted for 48 h, BAT mitochondrial GDP binding of C57BL mice decreased by 70%, while that of KKAY mice showed no change. Both acute exposure to cold and norepinephrine injections increased GDP binding in KKA^Y mice by 90% and 131%, respectively. These results indicate that low BAT thermogenesis in response to sucrose intake may be a cause of obesity in KKA^Y mice, and this may be brought about by defects in the central nervous system.

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Unsaturated long chain fatty acids are known to inhibit the binding between estrogen and estrogen receptor, or progesterone and progesterone receptor in rat uterus. The effects of long chain fatty acids on the binding between androgen receptor of castrated rat prostate and³H-R1881 were studied. The binding was not affected by saturated fatty acids such as palmitic acid (16: 0) or stearic acid (18: 0). But unsaturated fatty acids such as oleic acid (18: 1), arachidonic acid (20: 4) and docosahexaenoic acid (22: 6) inhibited the binding between androgen receptor and ³H-R1881. The inhibitory effect of arachidonic acid was dose dependent. Scatchard analysis showed that the addition of arachidonic acid markedly decrease the number of binding sites of androgen receptor. But the dissociation constant was not affected. The inhibitory effect of arachidonic was not a competitive one.

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A 36-yr-old man with multiple endocrine neoplasia (MEN) type I had an ectopic growth hormone-releasing hormone (GHRH) syndrome due to a GHRHsecreting pancreatic tumor. The immunoreactive (IR)-GHRH concentration in his plasma ranged from 161 to 400pg/ml (299±61pg/ml, mean±SD; normal, 10.4±4.1pg/ml), and a significant correlation was found between his plasma IR-GHRH and GH (r=0.622, p<0.02). After removal of the pancreatic tumor, the high plasma GH concentration returned to nearly the normal range (42.2±31.3 to 9.6±3.8ng/ml). These changes paralleled the normalization of his plasma IR-GHRH (16.1±3.8pg/ml) and some of his symptoms related to acromegaly improved. However, plasma GH (7.7±1.3ng/ml) and IGF-I (591± 22ng/ml) concentrations were high at 12 months after surgery, suggesting adenomatous changes in the pituitary somatotrophs.
Before surgery, exogenous GHRH induced a marked increase in plasma GH, and somatostatin and its agonist (SMS201-995) completely suppressed GH secretion, but not IR-GHRH release. No pulsatile secretion of either IRGHRH or GH was observed during sleep. An apparent increase in the plasma GH concentration was observed in response to administration of TRH, glucose, arginine or insulin, while plasma IR-GHRH did not show any fluctuation. However, these responses of plasma GH were reduced or no longer observed one month and one year after surgery.
These results indicate that 1) a moderate increase in circulating GHRH due to ectopic secretion from a pancreatic tumor stimulated GH secretion resulting in acromegaly, and evoked GH responses to various provocative tests indistinguishable from those in patients with classical acromegaly, and 2) the ectopic secretion of GHRH may play an etiological role in the pituitary lesion of this patient with MEN type I.

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Fibronectin (FN) has been considered to be involved in the malignant transformation of cells. It was thus of interest to morphologically study the cell surface distribution of FN in various types of thyroid neoplasms employing immunohistochemical techniques. Immunohistochemical staining using the specific anti-human FN antibody was performed by the avidin-biotin-peroxidase complex procedure. Compared to the staining pattern of normal thyroid tissues (control), differences in staining in malignant thyroid lesions and even in fetal and embryonal adenomas were evident, often on the lateral and basal aspects of the cell membrane. Colloid adenomas, however, showed FN staining on the basement membrane, basically similar to the controls. Moreover, in thyroid carcinoma, different staining patterns relating to each of the histological types were observed. The present investigation demonstrates the heterogeneity of the thyroid neoplasms in terms of FN binding.

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The serum BGP level was assayed in patients with hyperthyroidism (untreated and remittent cases) and hypothyroidism. The mean serum BGP concentration was 9.7± 0.90 ngml in 30 patients with untreated hyperthyroidism which was significantly higher than the 2.7± 0.38 ng/ml in 15 remittent patients and 1.3± 0.31 ng/ml in 13 patients with hypothyroidism (p<0.001, p<0.001). Serum BGP had a significant positive correlation with the concentrations of free triiodothyronine and alkaline phosphatase in the serum, while it had a significant negative correlation with serum PTH. In the patients with hypothyroidism, serum BGP increased significantly in parallel with increases in serum free triiodothyronine with thyroxine therapy. In the patients with hyperthyroidism, serum free triiodothyronine decreased significantly after the first month of methimazole treatment, and fluctuated within then ormalrange after two months. Serum alkaline phosphatase and BGP did not show significant changes during the first six months of treatment, although they were eventually reduced significantly at the end of one year.
These results suggest that thyroid hormone directly stimulates the synthesis and secretion of BGP in existent osteoblasts and also acts on the bone remodeling cycle, therapy accelerating the rate of bone formation; the latter action may occur over a long period.

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It was reported that unsaturated long chain fatty acids, such as arachidonic acid, oleic acid and docosahexaenoic acid inhibit the binding between progesterone and estrogen receptors and steroid hormones. Most of the long chain fatty acids are contained in phospholipids within the cells. The effect of phospholipids on the binding between R5020 and progesterone receptors was studied. Phosphatidyl ethanolamine and sphingomyelin had no effect on binding, but phosphatidyl inositol and phosphatidyl serine inhibited the binding 53% and 34% respectively. The effect of phosphatidyl inositol on the binding between R5020 and progesterone receptors was dose dependent. Scatchard analysis revealed that the addition of phospholipid markedly decreased the number of binding sites from 1398fmol/mgp to 258fmol/mgp, but the dissociation constant was little affected.

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Two postpartum and 7 spontaneous episodes of transient hypothyroidism developed during 5 years of observation in a florist with autoimmune thyroiditis and seasonal allergic rhinitis. The spontaneous episodes recurred twice a year regularly in spring and in autumn, the seasons of her allergic rhinitis. In most of these episodes as well as in the postpartum ones, hypothyroidism was preceded by transient thyrotoxicosis. In addition to the similarity in the clinical course, the changes in antimicrosomal antibody titers in pregnancyunrelated episodes were almost identical to those in postpartum episodes. Throughout the observation periods, she had supranormal serum total immunoglobulin (Ig) E concentrations and high antigen-specific Ig E levels for various pollen extracts such as cedar and ragweed. However, we failed to demonstrate a correlation between thyroid function and total or any antigen-specific Ig E level.
The present findings suggest that the pregnancy-unrelated thyroid dysfunction in our patient developed as a result of silent thyroiditis and through similar immunological mechanisms to those in postpartum thyroiditis. And it is suspected, though unproved, that the seasonal recurrence of pregnancyunrelated thyroiditis has some causal relation to her allergic disease.

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A reversed-phase high-performance liquid chromatographic method for the simultaneous determination of cyproterone acetate (CPA), 15β-hydroxycyproterone acetate (15β-OH-CPA) and cyproterone (CP) was reported. This method was specific, sensitive, precise, easy and rapid for determination of the serum concentrations of these steroids in patients receiving CPA. Although no peak corresponding to CP was observed for serum, peaks corresponding to CPA and 15β-OH-CPA were detected and well separated in all subjects undergoing long-term CPA therapy. In these patients, there seemed to be a dose-dependent relationship between the amount of CPA administered and the serum concentrations of these steroids, and the serum concentrations of CPA were either similar or low compared with those of 15β-OH-CPA.
In conclusion, this simplified method is thought to be very valuable for studies on the pharmacokinetics of CPA and 15β-OH-CPA, and on the relationship between the CPA dosage and the therapeutic or side effects on adrenal and gonadal steroid production.

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Very little has been known of the biochemical function of a human adrenocortical carcinoma cell line, SW-13. In this study, the production of several adrenal steroids and 3', 5'-cyclic adenosine monophosphate (cAMP) were investigated in this cell line. The cells were iucubated in L-15 medium containing 0.1% bovine serum albumin with several reagents in an atmosphere of 5% CO₂ and 95% air for 2 hours at 37°C. Aldosterone (Ald), corticosterone (B), cortisol (F), dehydroepiandrosterone sulfate (DHEA-S) and cAMP were simultaneously assayed by specific radioimmunoassays in the medium and cells. Significant increases in cAMP production were observed by cholera toxin (10ng/ml) and forskolin (10nM), both direct stimulators of adenylate cyclase, in the cAMP concentration without an increase in the steroids. The DHEA-S concentration in the medium was significantly increased by angiotensin-II (10^-7M), noradrenalin (3×10^-5M), adrenalin (3×10^-5M) or α-melanocytestimulating hormone (α-MSH, 10^-7M), none of which was associated with cAMP production. Neither adrenocorticotropin (10^-10M) nor human chorionic gonadotropin (500mIU/ml) stimulated the release of the steroids or cAMP production. A calcium ionophore, A23187 (10^-7M), and 12-O-tetradecanoylphorbol-13-acetate (10^-8M), a direct stimulator of protein kinase C, stimulated the release of DHEA-S, but not those of Ald, B and F. The results suggest that SW-13 retains functioning adenylate cyclase which, however, is not linked with steroidogenesis and that DHEA-S is produced probably by the mechanisms which involve protein kinase C system or calcium ion. This report provides the first demonstration of cAMP and DHEA-S production in SW-13 and suggests that this cell line is potentially useful for investigating the mechanisms of steroidogenesis in the human adrenal cortex.

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We examined a case of primary hypomagnesemia with associated hypocalcemia and hyperphosphatemia. It was found, on treatment with magnesium, that there was a significant negative correlation between the serum magnesium level and the percent tubular reabsorption of phosphate, especially when the serum magnesium concentration was above 1.0mg/dl, in the patient. It is suggested that the serum magnesium concentration might play an important role in urinary phosphate excretion, probably in relation to the parathyroid hormone function.

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A 41-year-old man with acromegaly was suffering from chronic, progressive backache and aware of reduction in his body height. Endocrine studies revealed increased glucose non-suppressible serum growth hormone (GH) and serum prolaction (PRL). Pituitary microadenoma was detected by a computerized axial tomogram and subsequently resected by trans-sphenoidal adenomectomy. The tumor proved to be a mixed GH- and PRL-secreting adenoma by electron microscopy and immunoperoxidase staining. Concurrent investigation of backache and reduced height disclosed markedly reduced radiodensity of the spinal bones, bilateral nephrocalcinosis, and hypercalciuria, which were ascribed to renal tubular acidosis (RTA) demonstrated by reduced urinary excretion of acids and insufficient reduction of urinary pH following oral administration of ammonium chloride. From the analogy to certain endocrinopathies, it appears likely that enhanced calcium metabolism and resultant hypercalciuria due to excses GH and PRL have led to the development of RTA, which further enhanced calciuria. Such enhanced calcium metabolism and consequent hypercalicuria conceivably led to accelerated demineralization of the spine and resulted in the reduced height of this patient in his early forties.

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A sensitive sandwich enzyme immunoassay for human growth hormone (hGH) using monoclonal antibody is described. A monoclonal anti-hGIl IgGcoated polystyrene ball was incubated with hGH and subsequently with affinity-purified rabbit anti-hGH Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorimetry using 3-(4-hydroxyphenyl) propionic acid as a substrate. The detection limits of hGH in serum and urine were 1.5 ng/l using 20μl of serum and 0.2 ng/l using 0.15ml of urine, respectively. The specificity and assay precision were satisfactory. hGH levels in serum and urine determined by the present sandwich enzyme immunoassay using monoclonal anti-hGH IgG-coated polystyrene balls were well correlated to those determined by the previous sandwich enzyme immunoassay using rabbit anti-hGH IgG-coated polystyrene balls. Levels of hGH in urine collected as first morning voids from healthy subjects aged 19-28 yr were 6.4±3.2 (SD) ng/g creatinine. However, the present assay gave lower hGH levels than the previous assay. This was at least partly explained by the fact that hGH in urine was less efficiently bound to monoclonal anti-hGH IgG-polystyrene balls than standard hGH, while the binding of hGH in urine and standard hGH to rabbit anti-hGH IgG-coated polystyrene balls was equally efficient. In addition, gel filtration showed that 22K hGH, a major component, in urine was less efficiently bound to monoclonal anti-hGH IgGcoated polystyrene balls than standard 22K hGH. The nature of hGH in serum and urine remains to be investigated.

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Leuprolide, a synthetic LHRH analog, inhibited growth of the Dunning R 3327 androgen-sensitive rat prostatic tumor and induced weight loss in male accessory sex organs. The relationship between the mode of administration and efficiency of the treatment was examined. Maintenance of the drug level in vivo seemed to be one of the important factors in the suppression of tumor growth, while a decrease in the weight of the accessory sex organs was mainly dependent on the dose administered. No treatment with leuprolide surpassed the effect caused by castration. Cytosolic androgen receptor and acid phosphatase activity in the tumor tissues were not changed significantly after treatment with leuprolide.

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