The existence of insulin receptors in rabbit erythrocytes was studied by evaluating the specific binding of ¹²⁵I-insulin to erythrocyte membranes. The binding of ¹²⁵I-insulin was pH, time and temperature dependent. Maximal binding was achieved by incubation for 20hr at 0°C. The optimum pH was 7.4. Treatment with cations and enzymes enhanced the specific binding except for with trypsin, the treatment which greatly reduced the binding. Unlabeled insulin over a wide range of concentrations competitively inhibited the binding of ¹²⁵I-insulin, while the binding was little affected by structurally unrelated hormones. Scatchard plot was represented as a concave curve. Binding sites of relatively high affinity (K₁=0.9×10⁹M^-1) and low capacity (8.0×10¹³/g protein) could be distinguished from those of lower affinity (K₂= 0.8×10⁷M^-1) and higher capacity (1.8×10¹⁵/g protein). Hill's analysis and dissociation of ¹²⁵I-insulin from membranes demonstrated the characteristics of negative cooperation between receptor sites.
Both incorporation of H₃³²PO₄ to erythrocyte membranes and uptake of ⁴⁵Ca were significantly reduced by the addition of unlabeled insulin. Unlabeled insulin produced no effect on uptake of ⁴⁵Ca into trypsin-treated erythrocytes.
On the basis of these results, it was suggested that rabbit erythrocytes might possess biologically significant insulin receptors located on the cell membranes.

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The differentiation of the folliculo-stellate (F-S) cells was electronmicroscopically investigated in the normal male adult rats from the Wistar, Wistar-Imamichi, Holzmann, Spraque-Dowley and Donryu strains. The F-S cells may be divided into the five types according to the granulation. Each type is, however, provided with the common characteristic features, i. e., the stellate shape due to projecting the cytoplasmic processes and a tendency to embrace an acidophil. The first type is an agranular independent or anastomosing immature cell. It is different in shape and arrangement from the follicular cell, but similar in agranularity and immaturity to it. The second is a slightly differentiated cell, in which scanty small secretory granules 50-100nm in diameter begin to appear near the plasma membrane. The third is a moderately differentiated cell providing the regularly row arrangement of secretory granules 100-200nm in diameter along the plasma membrane, corresponding, in fine structure, with a corticotroph. The fourth is a fully differentiated cell with heavy granulation, whose secretory granules 150-250nm in diameter are accumulated in the whole cytoplasm, suggesting the storing type. It is difficult to determine whether the fourth type coincides with a hypergranulated corticotroph or a stellate thyrotroph. The fifth is a kind of fully differentiated cell which may refer to the releasing phase of the fourth type, being characterized by the dispersion or loss of minute secretory granules of low density as large as50nm in diameter, associated with the cored vesicles. The population density of the above five types increased in the sequence, 5th→4th→2nd→1st→3rd type in the gland. Namely, the 3rd (corticotroph) type and 1st (agranular) type are predominantly distributed, and the5th (releasing) type and 4th (hypergranulated) type are rarely.

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A small yet significant increase of immunoassayable pancreatic somatostatin concentration (0.107±0.005 vs. 0.156±0.017μg/g at 24hr, p<0.05) was found in rats, 24hr as well as 7 days after treatment with a diabetogenic dose of streptozotocin (65mg/kg BW). These animals were characterized by marked decreases of insulin in the pancreas without any significant changes in pancreatic glucagon concentration. These results suggest that an abrupt deprivation of insulin from islets results in an elevation of pancreatic somatostatin concentration, and that glucagon in the pancreas plays a minor role in determining pancreatic somatostatin concentration in rats with insulin-deprived diabetes of short duration.

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Studies of the effect on feedback mechanisms in nycthemeral fluctuation of plasma TSH and of its sex-related variation were carried out on normally fed (Purina) and propylthiouracil (PTU) treated rats. Plasma TSH concentration of purina fed rat was significantly higher in males than in females (M=96±14, F=49±3μu/m/I, P<0.01). Plasma T4concentration also showed a high tendency in males compared to females (M=7.4±0.5, F=5.8±1.0μg/dI, 0.05<P<0.1) but the statistical difference was not significant.
Plasma TSH demonstrated a typical periodicity characterized by a zenith at 1200hr and a nadir at1800hr as in the previous reports. Plasma TSH concentrations rose apparently after the diet was switched to the one containing 0.15% of PTU. On the other hand, plasma T4concentrations decreased to the very low levels following PTU administration. Concerning to the nycthemeral fluctuation and its sex related variation following PTU administration, nycthemeral fluctuation in male rats disappeared rapidly, whereas it was preserved in female rats. These facts showed that it kept more persistent fluctuation in female rats than in male rats under the high plasma TSH levels. Furthermore, female rats showed higher concentration of plasma TSH after PTU treatment compared to male rats.
These results suggest that the nycthemeral fluctuation of plasma TSH was abolished or masked by the feedback mechanism of hypothalamo-pituitary-thyroid axis and there was a sex-related difference.

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Effect of the infusion of acetylcholine on the secretion of gut glucagon immunoreactivity (gut GI) that was measured using C-terminal specific glucagon antiserum after pancreatectomy, and gut glucagon-like immunoreactivity (gut GLI) that was obtained by subtracting GI from total glucagon-like immunoreactivity (total GLI) which was measured using non-specific glucagon antiserum, was investigated in sixteen pancreatectomized dogs untreated with insulin, in order to demonstrate whether the secretion of gut GI and gut GLI is influenced by the parasympathetic nervous system.
During the infusion of acetylcholine at a rate of 10μm/kg/min, gut GI in the femoral venous blood showed a significant increase from the basal value of 181±22pg/ml to a maximum of 569±107pg/mI at 30min (p<0.01), and “true gut GI secretion increment” in the portal venous blood showed a maximum significant increase of 916±144% at30min from the basal value (p<0.001). However, gut GLI showed no significant change. One shot administration of atropine at a rate of 15μg/kg could significantly inhibit the stimulatory effect of acetylcholine on gut GI (p<0.05-0.001).
It is concluded that the parasympathetic nervous system might play an important role in the control mechanism of the release of gut GI, but not of gut GLI in pancreatectomized dogs untreated with insulin.

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Ovariectomy performed at mid pregnancy increased the specific binding of ¹²⁵I-prolactin to the mammary gland (expressed as cpm/mg DNA) in rats 40hr after the operation. When adrenals were removed simultaneously at the time of ovariectomy, no increase in the specific binding was observed. An appreciable amount of lactose was found in all mammary glands of ovariectomized-adrenalectomized animals, though the content was less than half of that of the ovariectomized animals. The plasma level of prolactin in ovariectomized and ovariectomized-adrenalectomized rats was slightly higher than that in sham operated control rats, though the differences were not statistically significant. These results seem to indicate that the adrenal gland is necessary for the induction of the site of specific binding of prolactin in the mammary gland of the rat and that an increase in the circulating levels of prolactin does not seem to play an essential role in the initiation of milk synthesis in this species.

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Pituitary stellate cells from the normal adult male rats were immunohistochemically investigated at the light microscopical level by the use of rat TSH-β, porcine ACTH1-39, porcine ACTH17-39, rat FSH and ovine FSH antisera. They were characterized by the stellate shape and a mimic engulfment of acidophils. In the present study, they were identified to be the ACTH cells but some were TSH cells. Although most of the corticotrophs showed a peripheral fringe immunostained with the porcine ACTH^17-39 antiserum, some others were stained diffusively throughout the cytoplasm. The latter cells coincided, in shape and in homogenous stainability of the cytoplasm, with the stellate TSH cells. Both cells did not correspond but were independent in distribution at the same site of the gland on the adjacent two sections. The stellate type of FSH cells could react with the ovine FSH antiserum, but not with the rat FSH antiserum. Absorption tests of the ovine FSH, procine ACTH^1-39 and procine ACTH^17-39 antisera were carried out by an application of procine ACTH. In consequence, the porcine ACTH^1-39 and porcine ACTH^17-39 antisera were absorbed efficaciously by the ACTH antigen at the dose of 10μg/ml, but the ovine FSH antiserum was not enough absorbed by ACTH in the doses of less than 1mg/ml. It was not finally concluded whether or not the single stellate cells produced ACTH and FSH.

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Chlormadinone acetate was bound to cytosol from the human benign prostatic hypertrophy in a high affinity fashion. The kd and number of maximum binding site of the binding were 5.4±0.7×10^-9M and 67.9±5.8fmol/mg protein, respectively. When compared with the binding to dihydrotestosterone, the Kd for chlormadinone acetate was greater. The number of maximum binding site for chlormadinone acetate was observed to be smaller than that for dihydrotestosterone, but these two values were not different statistically. The binding of chlormadinone acetate was inhibited significantly by the addition of R1881or cyproterone acetate. However, dihydrotestosterone revealed itself to be a weak inhibitor of the binding. The cytosol prelabelled with chlormadinone acetate was not bound to the nuclei of the human prostate.

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The present report investigated steroidogenesis in vitro in testis tissues obtained from two boys aged8months and4years with ambiguous external genitalia and male vagina, and a 4-year-old boy with true precocious puberty. Histologically, testes of the former two boys are still immature and the testis of the last one contains differentiated Sertoli cells and primary spermatocytes, but no mature Leydig cells are recognized in any of them. In each testis, 17β-hydroxysteroid oxidoreductase is active for androstenedione in the presence of an excess amount of NADPH, while Δ5-3β-hydroxysteroid dehydrogenase and Δ4-steroid 5α-reductase activities are limited.17β-Hydroxylase and C17-20 lyase are significantly active in each testis and are enhanced in the testis of the boy with precocious puberty. Although the testis tissue used in the present study may not be biologically normal and the number of cases investigated is still limited, the above results indicate that active 17β-hydroxysteroid oxidoreductase is present in immature human testes and that Δ5-3β-hydroxysteroid dehydrogenase may become active in the human testis at the advanced stage of the development of testicular function during the puberty.

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Plasma active and acid activated inactive renin was measured in healthy subjects and in patients with diabetes mellitus. The angiotensin I generated from the incubation of non-acidified plasma with pig renin substrate was expressed as plasma renin concentration (PRC) and that from acidified plasma was expressed as total renin concentration (TRC). The inactive renin concentration (IRC) was calculated as TRC minus PRC. With regard to plasma renin activity (PRA) and PRC, no significant difference was found between normal and diabetic groups. TRC and IRC in diabetics with no clinical sign of microangiopathy were22.8±1.7and15.2±1.3 ng/ml/h (mean±SE), and these values were not significantly different from those in the healthy subjects (20.5±1.5and13.2±1.5ng/ml/h). However, TRC and IRC in diabetics with retinopathy and clinical nephropathy was38.8±3.4and30.7±3.2ng/ml/h, and these values were significantly higher than those in the above two groups, respectively. Moreover TRC and IRC in diabetics with retinopathy and no clinical nephropathy was33.8±5.7and24.9±5.5ng/ml/h, and these values were significantly higher than those in the control group. IRC was not significantly correlated with fasting blood sugar and mean blood pressure levels, however a significant correlation was found between IRC and BUN, and IRC and P. S. P. excretion in 15 minutes.
These findings suggest that increased inactive renin in diabetes mellitus may be related to the progression of the renal lesions associated with diabetic microangiopathy.

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The antiandrogenic properties of a new nonsteroidal antiandrogens, AA560 (N-(2-chloromethyl-2-hydroxypropionyl)-3, 4, 5-trichloroaniline) were investigated. The ventral prostate, dorselateral prostate and coagulating gland weights in rats given AA560 at1-9mg/head were significantly less than those in the intact rats. The seminal vesicle weights in rats given3-9mg/head were significantly less than those of the intact group. In intact animals given daily3or9mg of AA560there were significantly increases of serum FSH, LH and testosterone concentrations.
In the in vivo experiment, the pretreatment with AA560decreased the uptake of ³H-androgens in the nuclear fraction of the ventral prostate. On the other hand, a significant increase in the uptake of³H-radioactivity in the cytosol fraction was observed.
It was proved by the in vitro displacement study that AA560inhibited the binding of 5α-dihydrotestosterone with a receptor protein in the prostatic cytosol.

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The role of hypophysis in the regulation mechanism of the secretion of gut glucagon immunoreactivity (gut GI) that was measured using C-terminal specific glucagon antiserum after pancreatectomy, and gut glucagon-like immunoreactivity (gut GLI) that was obtained by subtracting GI from total glucagon-like immunoreactivity (total GLI) which was measured using non-specific glucagon antiserum, was investigated in depancreatized dogs.
Plasma glucose, gut GI and gut GLI levels were found to increase in totally depancreatized dogs. The former two showed a significant decrease after hypophysectomy, and were reversed by the hypophysis-transplantation, while gut GLI was not affected either by hypophysectomy or hypophysis-transplantation. Intramuscular injections of human growth hormone (HGH) or adrenocorticotropic hormone-Z (ACTH-Z) to depancreatized-hypophysectomized dogs had no effect on plasma glucose level or gut GI.
It is concluded that hypophysis may promote the secretion of gut GI after pancreatectomy, but not of gut GLI. Gut GI seems to regulate plasma glucose level after pancreatectomy. However, the precise regulation mechanism of gut GI by the hypophysial hormone after pancreatectomy is not clarified yet.

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A single oral dose (300mg) of γ-oryzanol extracted from rice-bran oil produced a significant reduction on the elevated serum TSH levels in hypothyroid patients. Similarly, chronic treatment with γ-oryzanol resulted in decreased serum TSH levels in 6 of 8 patients. There was no change in the serum levels of thyroxine-iodine and triiodothyronine during the study. In addition, there was no difference in the serum TSH response to TRH in hypothyroid patients and normal subjects.
These observations suggest that γ-oryzanol inhibits serum TSH levels in patients with primary hypothyroidism, possibly by a direct action at the hypothalamus rather than the pituitary.

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After a single PMS (50IU) injection to 25-day-old rats, FSH receptor content of the ovarian tissue increased progressively for4days, then showed a tendency to decrease, while LH receptor content remained unchanged for 3 days, then gradually increased. From these facts, we established a radioreceptor assay system, employing 3, 000rpm precipitates of homogenates of the ovaries obtained3days after PMS injection as the receptor preparation. The dissociation constant of the binding of the receptor preparation to rat FSH was 7.2×10-11M. The standard curve was obtained with 0.125-16ng/tube of NIAMDD rat FSH1-3. Purified preparations, NIAMDD rat LH I-4 and NIAMDD rat TSH I-4influenced the binding only at high concentrations possibly owing to FSH contamination. When the anterior pituitary homogenates obtained from rats in the various physiological states were assayed by this system, the intra-assay coefficient of variation and inter-assay coefficient of variation were 7.5 % and13.7%, respectively, and the assay values were well correlated with those obtained by radioimmunoassay (r=0.988, the slope of the regression line=1.14).

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The effect of propranolol treatment (60mg per day, three days) on glucose disposal rate (K-value) was investigated in nine patients with hyperthyroidism. K-value was improved in4cases and aggravated in5cases. The fasting levels of plasma free fatty acids (FFA) before the administration of propranolol in the improved cases were significantly higher than those in the aggravated cases. The propranolol treatment markedly reduced FFA levels only in the improved cases. These results suggest that the impaired glucose tolerance frequently seen in hyperthyroidism patients could be partly attributed to the increased level of plasma FFA.

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Several binding characteristics of the Dexamethasone (DEX)-receptor complex (RC) to nuclei from rat tissues were examined in the presence of Ethidium Bromide (EB). In the cell-free experiments, EB partially prevented the binding of the liver DEX-RC to nuclei and two kinds of nuclear binding of the DEX-RC, one was sensitive and the other was resistant to EB, were observed. In addition, the EB-resistant nuclear binding that showed a high-affinity and low-capacity nature was observed only in the bindings of the³H-DEX-RC to nuclei derived from the liver of fetal and adult rats and the thymus of adult rats. The bindings of³H-5α-Dihydrotestosterone (DHT)-RC and³H-17β-Estradiol (E₂)-RC to liver nuclei exhibited low-affinity and non-saturation even in the presence of EB. Furthermore, it was revealed that the EB-resistant sites of the nuclear binding were for the “DNA-unbound” form of³H-DEX-RC, while the RC of “DNA-bound” form was bound to the EB-sensitive sites.

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The mechanism and specificity of product activation of17ß-hydroxysteroid oxidoreductase for interconversion of androstenedione and testosterone were investigated using cell-free homogenates of human testes as an enzyme preparation.
The reactions of oxidation and reduction catalyzed by the enzyme followed the Michaelis-Menten formula. Addition of testosterone to the incubation medium resulted in increased Vmax and decreased the Km of reductase function of the enzyme. Androstenedione added to the incubation medium increased both Vmax and Km of the enzyme for the oxidase function.
The apparent Km of the reductase function for [³H] androstenedione as a substrate in the presence of a fixed concentration of [¹⁴C] testosterone was found identical with the apparent Kd for the androstenedione as an effector (activator) for the oxidase function of the enzyme calculated from the simultaneous increase of [¹⁴C] androstenedione produced from the fixed concentration of [¹⁴C] testosterone. Stmilarly, the apparent Km of the oxidase function for [³H] testosterone as a substrate in the presence of a fixed concentration of [¹⁴C] androstenedione was identical to the apparent Kd for the testosterone as an effector (activator) for the reductase function of the enzyme calculated from the simultoneous increase of [¹⁴C] testosterone produced from the fixed concentration of [¹⁴C] androstenedione.
None of the free17-oxo and17β-hydroxy steroids examined showed substrate activation or product inhibition of the enzyme except for androstenedione which showed product inhibition at high concentration.
The present findings in combination with our previous data suggest the presence of two sites on the17β-hydroxysteroid oxidoreductase of the human testis, one specific for androstenedione and the other specific for testosterone, each serving as an active site for the substrate and also as an activation site for the other active site.

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The concentration of luteinizing hormone-releasing hormone (LH-RH) in both cerebrospinal fluid (CSF) and plasma in 36 women with various conditions were determined, and the correlation of LH-RH concentrations between plasma and CSF was studied. Paired samples of plasma and CSF were obtained just before operation and were extracted with methanol for LH-RH: radioimmunoassay. LH and FSH were also measured by radioimmunoassay. The mean plasma LH-RH levels were 3.79pg/ml in the follicular phase, 4.15pg/ml in the luteal phase, 12.73pg/ml in postmenopausal women and 2.31 pg/ml in pregnant women at term. No immunoreactive LH-RH in the CSF was found in 29 out of 36 subjects, and the remaining 7 subjects showed very low levels (less than 0.95 pg/ml). The LH-RH levels in the CSF of post-menopausal women were not higher their those in the remaining 3 groups, although their plasma levels of LH-RH were significantly (p<0.001) elevated. The mean plasma levels of LH and FSH in post-menopausal women were significantly higher than those in normal cyclic women and in pregnant women. The present results suggest that little or no LH-RH is present in the CSF, and it is not clear that there is any correlation between the LH-RH level of plasma and those of CSF.

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