It has been established that insulin is synthesized as proinsulin within the pancreatic beta cell and then secreted together with connecting peptide, or C-peptide, which forms the major portion of the link between the insulin A and B chains in the proinsulin molecule. And it is generally accepted that C-peptide, unlike insulin, is not degraded by the liver. It is impossible to measure the serum insulin concentrations by the usual method of radioimmunoassay in subjects treated with exogenous insulin. While C-peptide is specific in its structure to species so that it is possible to measure its concentrations in such subjects. To assess the effect of pregnancy on insulin secretion by the pancreatic beta cell and its metabolic clearance, the following studies were performed during mid and late pregnancy and also 1 week and 4 weeks post partum. Glucose, insulin and C-peptide concentrations were measured during basal fasting conditions and following the administration of oral glucose or intravenous arginine. According to the recommendatory criteria of the Japan diabetic society, the carbohydrate metabolic function was classified as normal, borderline and diabetic.
During late pregnancy, the mean value ± SEM for serum IRI concentrations after an overnight fast had a tendency to decrease in diabetic (8.6 ± 0.7 μU/ml) as compared to normal (10.4 ± 0.9 μU/ml). Fasting values in any group during pregnancy had a tendency to increase against the respective groups of non-pregnant women. During 50 g oral glucose tolerance tests (OGTT) in normal, it reached a peak value of 45.1 ± 3.8 μU/ml at 30 min. The total area under the insulin concentration curve was the largest in borderline followed by normal and diabetic.
The fasting serum CPR in normal during late pregnancy was 2.56 ± 0.16 ng/ml and peaked at 7.52 ± 0.58 ng/ml 90 min. after an oral glucose administration. Then it fell to 4.63 ± 0.28 ng/ml at 180 min., which was higher than the fasting values. In borderline and diabetic, the fasting values for CPR were 2.11 ± 0.14 ng/ml and 2.10 ± 0.14 ng/ml, respectively. The area for CPR was the largest in borderline followed by normal and diabetic which was similar to the results for IRI. These results indicate that the response pattern of CPR during OGTT paralleled that of IRI in carbohydrate metabolic function. And also CPR response was slower in the rate, as compared to IRI.
It was possible to measure CPR concentrations in insulin-treated diabetic pregnant women with the usual method of RIA. The measured response of CPR during OGTT in these patients was the slowest and weakest.
The molar ratios of C-peptide to insulin were calculated. During pregnancy in normal, the ratio was 10.2 ± 1.8 in fasting and fell to the lowest values of 5.3 ± 0.5 at 30 min. after glucose administration, which tended to be higher than that of non-pregnant women at any time of OGTT.
A significant correlation was found between serum IRI and CPR (r=0.763, p<0.001) during OGTT in pregnancy as well as in non-pregnancy.
The incremental ratios of serum IRI and CPR to plasma glucose from a state of fasting to 30 min. after glucose administration were the highest in normal and the lowest in diabetic in both pregnant and non-pregnant women.
From mid to late pregnancy, the levels of CPR during OGTT were significantly elevated, while those of IRI were slightly and not significantly elevated. At 1 week after delivery, both IRI and CPR levels were significantly decreased, particularly in IRI, as compared to late pregnancy. From 1 week to 4 weeks after delivery, IRI was slightly but significantly elevated despite no change for CPR.
During 30 g arginine infusion tests for 30 min. in pregnancy, IRI had two peaks at 5 min. and 30 min. While CPR was raised rapidly after the start of the infusion, it sustained high levels for 30 min. without fractionation.

抄録全体を表示

The binding characteristics and physical properties of the nuclear receptors of rabbit liver were studied. The receptors were extracted with 0.4M KCl from purified hepatic nuclei and were incubated with increasing doses of¹²⁵I-triiodo-L-thyronine (L-T3) and stable L-T3 for 2 hours at 20°C. Scatchard analyses indicated that the association constant (Ka) of the nuclear receptors was 1.5×10¹⁰M^-1, and that maximal binding capacity was about 0.12 pmol T3 per 1 ml of nuclear extract, equivalent to 1 g of liver or 0.2 pmol T3 per mg of protein of the nuclear extracts. The nuclear binding sites were specific for L-T3. When compared by the molar concentrations of hormone analogues required to produce 50% inhibition of L-T3 binding, the relative binding affinities of triiodothyroacetic acid (Triac), D-T3, L-T4 and D-T4 were, respectively, 1/2, 1/4, 1/50 and 1/170 of L-T3. The binding affinity of Triac for the isolated hepatic nuclei was also nearly half that of L-T3. Furthermore, to displace radioactive Triac from the binding sites, Triac was half as effective as L-T3.
The molecular weight of the nuclear receptors was estimated to be about 40000-45000 by the elution profiles from a Sephadex G-100 column, and the sedimentation coefficient was slightly less than 4S on sucrose density gradient centrifugations. Both elution profiles from DEAE- and ECTEOLA- cellulose columns with a linear KC1 gradient showed a sharp, narrow peak of radioactive T3 at about 0.15-0.2MKCl.
The results obtained with rabbit liver were similar to those reported previously in rat liver except for the relative binding affinity for Triac. It is possible that this discrepancy may be due to a species-related difference.

抄録全体を表示

The effects of triiodothyronine (T3) on binding characteristics of nuclear T3-receptors were examined in rats. The T3-receptors were extracted by 0.4M KCl from the liver nuclei isolated from the following 3 groups of rats : (1) thyroidectomized, (2) thyroidectomized and treated with 230 ng T3/100 g body weight/day for 3 days, and (3) thyroidectomized and treated with 40 μg T3/100 g body weight/day for 3 days. Their association constant (Ka) and maximal binding capacity (Cmax) for T3 were determined by Scatchard analyses with and without correction for endogenous T3. The amount of endogenous T3 bound to the nuclear extracts was estimated on the basis of a specific activity of [¹²⁵I]-T3 injected 2 hours before sacrifice. It was demonstrated that Cmax values corrected for the endogenous T3 were 2.5 times greater in severely hyperthyroid than in hypothyroid rats. By contrast, corrected values for Ka remained unchanged in all 3 groups of rats, although uncorrected values were apparently decreased in the severely hyperthyroid rats. Validity of the correction was supported by the in vitro experiments preincubated with stable T3. The yields of nuclear protein-¹²⁵I-T3 complex in the procedure of extraction and the contents of DNA per g of liver were nearly the same in the 3 groups. The increase in Cmax of the nuclear receptors was directly related to mitochondrial α-glycerophosphate dehydrogenase activity. The results obtained indicate that the Cmax of nuclear T3-receptors is increased in a severely hyperthyroid state, related to hormonal action, and that the correction for endogenous T3 is essential for an accurate estimation of Ka and Cmax.

抄録全体を表示

The dynamics of the induction of nuclear triiodothyronine (T3) -receptors and mitochondrial α-glycerophosphate dehydrogenase (α-GPD) were studied in rat liver after a single injection of a large amount of T3. The maximal binding capacity (Cmax) and association constant (Ka) of the nuclear receptors were determined by Scatchard analyses with and without correction for endogenous T3 measured by radioimmunoassay. It was demonstrated that the administration of T3 induced sequential increases in the number of nuclear T3-receptors and α-GPD activity in the liver. The nuclear receptors were rapidly increased to 2.5 times the hypothyroid level and thereafter decreased with a half-life of about 2 days. A parallel change in α-GPD activity was noted after a lag period. The total amount of extracted nuclear proteins in the liver was increased 3 days after the administration of T3. It seems likely, therefore, that the T3-induced increase in nuclear receptors is responsible, at least in part, for the induction of this enzyme. The possibility is also suggested that the nuclear receptors may be a nonhistone protein selectively synthesized in an early stage of the hormonal stimulation. Following the injection of T3, marked changes in apparent Ka were seen when no correction was made for the amount of endogenous T3 bound to the extracted nuclear proteins. With the correction, however, Ka remained the same despite the changes in the nuclear receptors and enzyme activity. The results obtained provide further evidence for the hormonal modulation of the nuclear receptors which is closely linked with the hormonal effect.

抄録全体を表示

The steroid-receptor assays in advanced or recurrent human breast cancer have recently become important as a method for the predictions of a therapeutic response to endocrine therapy. Estrogen receptor (ER) and progesterone receptor (PgR) were measured by sucrose gradient centrifugation. Breast cancers (109), benign mammary tumors (22) and normal mammary tissues (10) were examined.
The tissue powders were homogenized with a motor-driven, teflon-coated glass homogenizer in a 2 ml buffer of 0.01 M Tris-HCl containing 0.0015 M EDTA pH 7.4 per gram of tissue. The homogenate was then centrifuged at 105,000g for 60 minutes to obtain the supernatant. The protein concentration of the supernatant had to be adjusted about 1.2%. One-half ml of the supernatant was incubated with 1 pM ³H-Estradiol (98.5 Ci/mmol) or 1 pM ³H-R5020 (70-80 Ci/mmol) at 0°-4°C for 3 hrs, and then the free steroids and nonbound protein were removed by dextran-coated charcoal. Two hundred fifty of the cytosol was layered on a 5% to 20% sucrose density gradient solution and centrifuged at 40,000 rpm for 18 hrs.
After centrifugation, fractions were collected in 40 test tubes at the rate of five drops per tube.
One ml of Tris-HCl buffer was added to each fraction, and the protein density at 278 nm was measured with a spectrophotometer. Ten ml of scintillator (Nonione-Toluene-PPOPOPOP) was added to each vial, and the radioactivity was counted with a liquid scintillation spectrometer.
The results obtained were as follows :
1) Specific binding of ER was observed at the 8S and one of PgR was in the 8S region.
2) About 45% of human breast cancers were ER (+), and about 20% were PgR (+). The positive rate of PgR was lower than that of ER. As for benign mammary tumors, one out of 10 fibroadenomas and one out of 3 giant fibroadenomas were ER (+), and all of the normal mammary tissues were ER (-).
3) There was no difference between premenopausal females and postmenopausal females in the positive rate of ER and PgR, and the percentage was not related to clinical stage or status of lymphnode metastasis. As for blood type, there was no difference in the positive rates of ER and PgR among A, B and 0 types. In the AB type, ER and PgR were negative in all the examined cases.
4) In the positive rate of ER, papillotubular carcinomas tended to be a little lower than the other histological types, while in the binding sites of ER, medullary tubular carcinomas were higher than scirrhous carcinomas. As for PgR, medullary tubular carcinomas tended to be higher than the others.
5) In 10 cases, the occurrence of ER in primary tumors and metastatic or recurrent lesions was almost identical, and their binding sites were at almost the same level.
6) In 39 cases which measured both ER and PgR, all 8 cases of PgR (+) showed ER (+), and there was a close relationship between ER and PgR.
7) The relationship between the occurrence of ER and the clinical response to endocrine therapy was examined in 21 cases. Six out of 12 cases of ER (+) (50%) and one out of 4 cases of ER (±) showed a response, but 5 cases of ER (-) showed no response.
8) Endocrine therapy was carried out in 11 cases out of the 39 cases in which both ER and PgR were measured. The cases of both ER (+) and PgR (+) seemed to respond better than those of ER (+) only.

抄録全体を表示

Simultaneous measurement of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) and 18-hydroxycorticosterone (18-OH-B) in the peripheral plasma was carried out on normal subjects and in patients with adrenocortical disorders.
The mean plasma levels of 18-OH-DOC at 0800h in normal males and in the follicular and luteal phases of normal females were 8.2 ± 3.9 ng/100ml (Mean ± SD), 7.8 ± 2.6 ng/ 100ml and 11.5 ± 2.8 ng/100ml, respectively. The corresponding levels of 18-OH-B in normal males and in the follicular and luteal phases of normal females were 10.3 ± 4.2 ng/ 100ml, 12.4 ± 4.5 ng/100ml and 13.8 ± 4.1 ng/100ml, respectively. No differences between the sexes nor the phases of the menstrual cycle were confirmed.
ACTH stimulation increased plasma concentrations of 18-OH-DOC and 18-OH-B by 5.1 and 4.4 times respectively, while dexamethasone markedly decreased these 2 steroids. An upright posture increased these steroids significantly.
In patients with Cushing syndrome, plasma levels of these 2 steroids were rarely high in cases with adrenocortical hyperplasia and adrenocortical carcinoma, while they were usually within the normal range in adrenocortical adenoma. These 2 steroid levels were increased in primary aldosteronism, idiopathic hyperaldosteronism and congenital 17α-hydroxylase deficiency. They were decreased in Addison's disease and the salt-loosing type of congenital 21α-hydroxylase deficiency. Patients with congenital 21α-hydroxylase deficiency (simple form) showed elevated levels of 18-OH-DOC and normal levels of 18-OH-B.
In vitro production of 18-OH-DOC and 18-OH-B was studied by tissue slices of the normal adrenal cortex, adrenocortical carcinoma causing Cushing syndrome, aldosteronoma and nodular hyperplasia with hyperaldosteronism. In the normal adrenal cortex, the mean production rates of 18-OH-DOC and 18-OH-B were 31 and 26 ng/g tissue/hr, respectively. ACTH and angiotensin II significantly increased the production of both 18-OH-DOC and 18-OH-B. In adrenocortical carcinoma, the production of these steroids was markedly diminished and not stimulated with either ACTH or angiotensin II. Aldosteronoma tissue produced these 2 steroids 20-40 times that of the normal adrenal tissue and was significantly increased with the addition of ACTH and angiotensin II. Nodular hyperplasia with hyperaldosteronism produced much 18-OH-DOC and 18-OH-B, but did not respond to ACTH and angiotensin II.

抄録全体を表示