Molar pregnancy has been repeatedly reported to be associated with hyperthyroidism. After removal of the molar tissue the patient's hyperthyroidism disappears. This fact suggests that the molar tissue produces a specific thyrotropin. But the control mechanism of the thyroid function under such conditions remains to be clarified. To throw some light on this problem, the author measured the serum levels of thyrotropins (human chorionic thyrotropin : hCT and human pituitary thyrotropin : hTSH) and performed TRH infusion test and various thyroid function tests (T₃, T₄ ETR, PBI, BMR, T₃-RSU, radioiodine uptake and TBG) on the patients with hydatidiform mole and trophoblastic neoplasias.
In molar pregnancy, an abnormally large amount of hCT was demonstrated as compared with normal pregnancies of similar gestational age, and the serum levels of hCT found to be correlated with the hCG serum levels (P<0.01). hCT became undetectable nearly five days after molar evacuation, and the time required for the disappearance of hCT after molar evacuation was longer than that of normal delivery. The base line levels of serum hTSH were statistically indistinguishable among women with molar pregnancies and those with normal pregnancies and nonpregnant subjects. The TSH response to TRH infusion in cases of molar pregnancy was somewhat blunted as compared with those of normal pregnancy of similar gestational age. The mean net increase (_??_ TSH) to TRH stimulation was smaller than that of normal pregnant women, but the serum hCT level did not change after the TRH infusion either in molar or normal pregnancy.
From the above data, the following items could be extracted : the increase in thyrotropic activity in plasma of the patients with hydatidiform moles is due to the accelerated production of a kind of thyrotropin (hCT) by molar tissue like as in hCG; such hCT is independent of the pituitary TSH, and consequently of TRH stimulation; although it remains to be shown whether or not such hCT from hydatidiform moles is identical with hCT from normal trophoblast, the molecular size seems to be larger than hCT from normal trophoblast since the disappearance time is longer.
All patients with hydatidiform moles should have hyperthyroidism if judged by T₃ T₄, PBI, BMR and radioiodine uptake, but practically, these patients show little or no clinical symptoms of hyperthyroidism. Since T₄ levels correlated with serum hCT levels (P<0.01), it is obvious that the elevated thyroid function is due to the increased hCT produced by the molar tissue. But the degree of the clinical symptom is modified by steroid hormones which influence TBG-binding capacity and by other factors. The production and secretion of hTSH by the pituitary is also controlled by the “cross-auto feed-back mechanism” of hCT or the “negative feed-back mechanism” of free thyroid hormone which is stimulated by hCT.
As a conclusion, high serum hCT levels, high thyroid hormone levels, and modified clinical symptoms by increased binding capacity of TBG and by suppression of hTSH secretion by two feed-back mechanisms, are the characteristics of molar pregnancy. And, in place of hypothalamic-pituitary-thyroid axis, molar trophoblast-thyroid axis is proposed to be acting as a dominant regulating mechanism for thyroid function in molar pregnancy.
Some hydatidiform moles develop into choriocarcinoma or chrioademoma destruence. Changes in hormone production taking place during such developments have not been studied serially. According to the author's study, like as in hydatidiform moles, hCT is also secreted by these chorionic neoplasias, but serum hCT levels were lower than in moles. In these case, mild hyperthyroidism was noted and slight suppression of hTSH secretion was observed.

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The influence of change in the feeding regimen on the circadian periodicity of plasma corticosterone levels was studied in adult Wistar rats under various lighting conditions. Blood samples were taken by the tail vein sampling method, and 10-20 μl plasma was used for the determination of corticosterone by means of the protein binding method.
Under the diurnal change of light, access to food was restricted to 2 h (09 : 00-11 : 00) or 8 h (09 : 00-17 : 00) during the light period. The two h restriction of food caused a phase shift of adrenocortical activity 11 days and 14 days after the restriction in female and male rats, respectively. No phase shift was observed, however, throughout the experimental period of 21 days in the case of the 8 h restriction, although the 24 h pattern of plasma corticosterone levels became flatter.
Unblinded rats exposed to constant light (LL) manifested an abolition of the circadian rhythm of adrenocortical activity at the end of the 5th week of exposure to LL. The rhythm was promptly recovered, however, when access to food was restricted to 2 h (19 : 00-21 : 00). When 2 h feeding was repeated at 12 h intervals, an aperiodic 24 h pattern was observed throughout the experimental period of 5 weeks.
In blinded rats in LL, a 16 h phase shift of adrenocortical rhythm was observed at the 5th week after blinding. This free-running rhythm was not affected by a 2 h food restriction of one week, but after 3 weeks of restriction, the peak phase shifted to the beginning of feeding time. A 2 h feeding at 12 h intervals caused an ultradian rhythm of adrenocortical activity at the end of the 2nd and 3rd week, which was synchronized with the ultradian feeding rhythm. However, this ultradian adrenocortical rhythm disappeared at the end of the 5th week.
These results suggest that the feeding rhythm is a more dominant factor than the light-dark alternation in the establishment of adrenocortical rhythm in rats. The significance of the ultradian rhythm in rats was discussed.

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The pathophysiology of serum protein is not clearly understood, although various kinds of serum protein have been investigated in various diseases. It is considered that the cellular and the humoral immunities play important roles in the pathogenesis of hyperthyroidism and chronic thyroiditis. The purpose of this study is to clarify an aspect of the pathophysiology of hyperthyroidism, measuring the concentration of various kinds of serum protein in patients with hyperthyroidism.
The concentration of various kinds of serum protein, such as prealbumin (Pre), albumin (Alb), α₁ acidglycoprotein (α₁ AG), α₁ antitrypsin (α₁ AT), α₂ HS glycoprotein (α₂ HS), ceruloplasmin (Cp), haptoglobin (Hp), α₂ macroglobulin (α₂ Mc), β_IC/A globulin (C₃), β_IE globulin (C₄), C₃ proactivator (C₃PA), IgG, IgA and IgM was estimated by the single radial immunodiffusion method (SRID) in 25 cases of normal subjects (14 cases of male and 11 cases of female), 24 cases of untreated hyperthyroidism (5 cases of male and 19 cases of female) and a patient with malignant exophthalmus. C₃PA and C₃ activator (C₃A) were assayed by Laurell's electroimmunoassay method in 14 cases of normal subjects and 15 cases of untreated hyperthyroidism.
The results obtained were the following :
1. The mean values of the concentration of Pre and Tf were significantly lower (P<0.05), and those of Alb, C₄ and C₃PA (SRID) showed a tendency to be lower in hyperthyroidism than in normal subjects. The mean values of the concentration of α₁AT, Cp, C₃ and IgG were significantly higher in the former than in the latter (P<0.05).
2. The relationship between the concentration of various kinds of serum protein and some parameters representing thyroid functions (BMR, T₃RU, T₄, cholesterol and free thyroxine index) was examined in hyperthyroid patients. Pre and Alb were negatively correlated with T₃RU, and α₁ AT and Cp were positively correlated with T₃RU, T₄ and free thyroxine index. However C₃ and IgG did not correlate with such parameters.
3. The variations in the level of various serum protein after the administration of antithyroid drugs were observed in 12 cases of untreated hyperthyroidism. The serum levels of Alb and Tf showed a tendency to gradually increase, and those of α₁AT, α₂HS and Cp showed a tendency to gradually decrease. There was no definite change in the con-centration of the other serum proteins.
These results suggest that the decreased serum levels of Pre and Alb and the increased serum levels of α₁AT and Cp in hyperthyroidism are closely related to the elevated blood levels of thyroid hormones in this disease. Decreased serum level of Tf in hyperthyroidism may be concerned with hypochromic anemia, which was frequently observed in this disease, and with the acceleration of the protein catabolism by thyroid hormones. The elevated serum levels of IgG and C₃ are considered to have resulted from some factors which are dependent not on thyroid hormones but probably on immunological mechanisms.

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Adrenal nodular hyperplasia is a rare clinical condition, and its pathogenesis is still under debate.
Recently we encountered a patient with Cushing syndrome due to huge bilateral adrenal nodular hyperplasia.
A 51 y.o. male, pilot, had been healthy and rather fat (body weight 75 kg) until 1971 when he was noticed to have glycosuria and hypertension. Since then, despite antidiabetic therapy, he lost weight gradually reaching 61 kg. Since August 1975 general lassitude and hypodynamia have been associated. Because of recently onsetted severe anorexia and continuous fever of 38°C he was admitted to our hospital on Nov. 7, 1975.
On his admission, mild buffalo hump, muscular atrophy of extremities, left pleural effusion, facial edema and abdominal distension were observed. However, plethoric moon face and cutaneous striae were not seen. Laboratory findings demonstrated some inflammatory changes of unknown origin, and moderate hyperglycemia and hypertension. Serum K was 3.4 mEq/l. Loss of weight, bloody pleural effusion and elevated LDH (640 Wr U) were suggestive of malignancy, however, no malignant lesion was found.
Treatment by insulin, antibiotics and aldactone were started, and after 4 weeks he become afebrile and pleural effusion also disappeared completely. After cessation of aldactone, hypopotassemia (2.2 mEq/l) and clinical manifestations of Cushing syndrome became evident.
Endocrinological examinations revealed increased urinary 17-OHCS excretion (17.5-32.1 mg/day) and hypercortisolism with no diurnal change, while urinary 17-KS remained within normal limits. His adrenals did not respond to metopyrone and 8 mg dexamethasone, but an exaggerated response was seen by ACTH administration.
Plasma ACTH. which was reported after surgery, was 10 pg/ml. Adrenal scintigraphy by 131-I adosterol showed typical head light sign.
Through drip infusion pyelography and retroperitoneal pneumoroentogenogram, a tumor-like shadow in the left suprarenal region and another round shadow in the right infrarenal region were observed. There was some suspicious shadow in the right suprarenal region also, but this was not certain.
From these observations, ectopic ACTH secretion from a right lower abdominal tumor or adrenal nodular hyperplasia were considered. On February 18, 1976, bilateral adrenalectomy was performed. Both adrenals were greatly enlarged but well capsulated and had multinodular appearances. They weighed 141 g (l) and 85 g (r), respectively. The tumor-like mass in the right lower abdomen contained yellow pus and was an abscess.
Histologically, a tumor-like hyperplasia of the adrenal cortex was confirmed. Major cells had bright cytoplasma and were rich in lipid granules which are typical properties of glucocorticoid secreting cells. Besides, sporadic distributions of reticular and glomerular zone derived cells were also observed.
The postoperative course has been quite good, and 37.5 mg of cortisone acetate and 0.25 mg of decadron have maintained his health until Dec. 1976. No apparent rise of plasma ACTH or beta-MSH has been noticed.
Such a huge nodular hyperplasia exceeding 200 g has never been reported before, and this case seemed to represent advanced features of this condition. Compared with the data of 19 previously reported cases, adrenal nodular hyperplasia appears to have the following characteristics : long duration of the disease (1-45 years), increased urinary 17-OHCS but rather normal 17-KS, unresponsiveness to metopyrone (6/9) and 8 mg dexamethasone (12/14), responsive to exogenous ACTH (11/17), and low plasma ACTH (7/8). These characteristics are commonly seen in cases with large tumors, including this case. However, in cases with small tumor effectiveness of metopyrone and 8 mg dexamethasone, and sometimes elevated plasma ACTH are reported, suggesting the likelihood of simple adrenal hyperplasia. In a middle-sized case reported by Choi (15 g),

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Although it has been proposed that cyclic AMP is an intracellular mediator in the stimulatory effect of ACTH on steroidogenesis, and that the action of cyclic AMP is exerted through the activation of protein kinase, it still remains to be resolved whether cyclic AMP-dependent protein kinases present in adrenocortical cells bear any direct or indirect relationship to steroidogenesis.
My previous observation that cyclic AMP acts directly on rat adrenal mitochondrial fraction to stimulate its steroidogenesis suggests that cyclic AMP-dependent protein kinase might be present in mitochondria per se. The present studies were undertaken to examine whether cyclic AMP-dependent protein kinase activity and the existence of its substrate could be demonstrated in rat adrenal mitochondrial fraction.
Adrenal subcellular fractions were obtained from male Sprague-Dawley rats pretreated with dexamethasone. Protein kinase activity was determined in a 0.2 ml reaction mixture containing 4.1 mM phosphate buffer (pH 6.5), 10 mM magnesium acetate, 0.3 mM EGTA, 10 mM sodium fluoride, histone (0.2 mg/tube), ATP-γ-³²P (2.5 μM, 0.2~3 μCi/tube) and subcellular fraction, with or without cyclic AMP. Incubation was carried out at 30°C for 5 min., and trichloroacetic acid-precipitable radioactive phosphate was assayed.
Cyclic AMP-dependent protein kinase activity was detected in the mitochondrial fraction as well as in soluble and microsomal fractions. The possibility of contamination of the mitochondrial fraction by other fractions was checked by comparing enzyme activities of protein kinase and various marker enzymes, 21α-hydroxylase, glucose-6-phosphate dehydrogenase, β-glucuronidase and 11β-hydroxylase, in each fraction. Contamination of microsomal and soluble fractions was found negligible, although the lysosomal enzyme activity was to some extent detectable in the mitochondrial fraction.
In order to examine the ability of mitochondrial proteins to serve as substrates for mitochondrial protein kinase, mitochondrial fraction was incubated with ATP-γ-³²P and Mg⁺⁺ without histone. ³²P-phosphate incorporation into the mitochondrial protein was clearly demonstrated. When phosphorylated mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis, several radioactive peaks with a predominant peak were observed. Cyclic AMP stimulated incorporation of phosphate into several protein bands on the gel without significant effect on the major peak.
These findings suggest that cyclic AMP-dependent protein kinase is present in adrenal mitochondria, and that a small but significant amount of mitochondrial protein undergoes phosphorylation by endogenous protein kinase in response to cyclic AMP. Therefore, cyclic AMP, by acting directly on adrenal mitochondria, might regulate its metabolic processes including steroidogenesis, and thus might act as the intracellular messenger of ACTH action.

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The establishment of radioimmunoassay for somatostatin is important for the assessment of its role in terms of humoral regulation, especially in the hypothalamo-pituitary relationship. The anti-somatostatin was prepared by the immunization of rabbits with somatostatin-HSG (α) conjugate. The antibody had no cross-reactivity with TRH, LHRH, pituitary hormones, insulin, C-peptide, or glucagon, but was cross-reactive with Des (Ala¹, Gly²)-somatostatin, somatostatinamide, or Tyr¹-somatostatin. ¹²⁵I-Tyr¹-somatostatin was prepared by the lactoperoxidase method, and purified through a carboxymethylcellulose column chromatography. The assay procedure was performed by incubation in 0.1 M phosphate buffer with 0.14 M NaCl, 0.025 M EDTA-2Na, 0.5% BSA (pH 7.4) at 4°C. The free and bound form were separated with polyethylene glycol. The double antibody technique and the dextran-coated charcoal technique were also employed for the separation of free and bound forms, but we prefer the former technique because of easy handling and cheap costs. The concentrations of immunoreactive somatostatin detected by this assay were 0.6-50 ng/ml.
Immunoreactive somatostatin in the tissue was extracted by aceton. The dilution curve of immunoreactive somatostatin extracted from the rat brain was paralleled to the standard curve gained by synthetic somatostatin. The recovery rate was approximately more than 50%. The extracts from the rat brain were identified by synthetic somatostatin on Sephadex G-25 column chromatography.
These data indicated this assay system might be useful for research applications concerning immunoreactive somatostatin in the tissues.

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It has been reported that gut glucagon immunoreactivity (Gut GI) and gut glucagon-like immunoreactivity (Gut GLI) increase markedly day by day in total pancreatectomized dogs without insulin injections. However, the regulation mechanism of the release of Gut GI and Gut GLI has still not been completely resolved.
The present studies were designed to find a part of the regulation mechanism of the release of Gut GI and Gut GLI in connection with hypophysis and also to examine the relation between gut glucagon and blood sugar levels after pancreatectomy.
Eight healthy mongrel dogs, weighing 10-13 Kg, which had fasted for twelve hours, were used. Under Ketalar and also Ravonal anesthesia, total pancreatectomy of these dogs was performed through a midline abdominal incision. Thereafter, these dogs were maintained without insulin therapy for three days. On the fourth day, under Ketalar anesthesia, the hypophysis of these dogs was removed under the direct vision through the temporal approach. Then for one day after this operation, these dogs were kept without further insulin and other hormone injections. On the next day, hypophyses of other dogs were transplanted individually into the hypoderm of the heads of these dogs. As controls, ten other dogs were used. Then total pancreatectomy was performed. These dogs were kept under nontreatment for eight days. In all these dogs, blood was sampled through the polyethylene catheter inserted into the femoral vein in the fasting state early every morning for eight days. The blood samples were assayed for blood sugar by the glucose oxidase method, immunoreactive insulin (IRI) by the double antibody method, and immunoreactive glucagon (IRG) by the talc method : glucagon immunoreactivity (GI), by using antibody “K30” highly specific for pancreatic glucagon (Unger, Texas), and total glucagon-like immunoreactivity (total GLI) by using a nonspecific glucagon antibody “K4023” (Novo, Denmark). This difference between total GLI and GI was considered to be Gut GLI.
1) In the control experiment of only total pancreatectomy, blood sugar levels showed significant increases (P<0.001) from 107±7 mg/dl, the value before the operation, to 306±24 mg/dl on the day after the operation; thereafter, they increased gradually, rising to a maximum of 756±124 mg/dl on the eighth day. IRI differed less than 5 μU/ml (P<0.05~0.01) in all the days after pancreatectomy from 14±2 μU/ml, the value before the operation. GI rose significantly (P<0.001) from 70±6 pg/ml, the value before the operation, to 251±32 pg/ml on the second day after the operation and thereafter showed a gradual increase, rising to a maximum of 680±167 pg/ml on the eighth day. Gut GLI increased significantly from 175±7 pg/ml, the value before the operation, to 452±74 pg/ml (P<0.01) on the second day after the operation and to 648±50 pg/ml (P<0.001) on the third day; thereafter, it remained stable at about 650 pg/ml until the eighth day. (Fig. 1)
2) In the hypophysectomized dogs after total pancreatectomy, blood sugar levels decreased significantly (P<0.05) from 362±22 mg/dl, the value before hypophysectomy, to 278±24 mg/dl on the day after hypophysectomy, and to 255±21 mg/dl, still lower on the second day. GI showed a significant decrease (P<0.05) from 438±36 pg/ml, the value before hypophysectomy, to 305±46 pg/ml on the day after the operation and to 262±52 pg/ml on the second day. However, Gut GLI showed no significant changes after hypophysectomy. (Fig. 2)
3) In the dogs which received hypophysis-transplantation after hypophysectomy following total pancreatectomy, blood sugar levels increased significantly (P<0.05) from 255±21 mg/dl, the value before transplantation. to 359±35 mg/dl on the second day after transplantation.

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