The synergistic effects of dietary obesity produced by the feeding of a high fat diet and stress induced by electric shocks on glucose tolerance and glucose-induced insulin release from the perfused pancreas were investigated.
Male Wistar rats weighing 90-100g were fed ad libitum for 12 weeks either a control (50% Starch; C) or a high fat diet (40% Butter; F). Some of the rats on both diets received 100 electric shocks of 1 sec. duration in the stress session for 1 hour per day for the last 3 weeks of the experimental period. Low stress (LS) groups were shocked at a fixed time (Inter Shock Interval : 36 sec.). High stress (HS) groups were shocked at random (ISI : mean = 36 sec, 9-108 sec. variable). Non-stress (NS) groups were not given any shocks. Rats were killed at 24 hours after the final stress session. Under NS conditions, rats in the F-NS group gained a significant amount of weight and had normal levels of fasting plasma glucose and insulin but an impaired glucose tolerance (k = 3.49). Insulin release from the perfused pancreas in the F-NS group showed a delay in the initiation of release by the stimulation of glucose (16.7mM), but the total amounts of insulin released did not differ from that in the C-NS group. On the other hand, the levels of plasma 11-OHCS in the fed state were much more highly elevated in the HS group than in the LS group, which was not influenced by the high fat diet. The fasting levels of plasma glucose in the F-HS group (121 ± 7 mg/ 100 ml) were significantly higher than those in the C-HS group (101 ± 7 mg/100 ml) in spite of a normal insulin concentration in plasma. In contrast to the normal glucose tolerance in the C-HS group (k = 5.14), glucose tolerance in the F-HS group (k = 3.04) was impaired. Insulin release from the perfused pancreas in response to glucose in both diet group was not significantly altered under LS conditions. In the C-HS group, however, the total amount of insulin released in the second phase was enhanced to 165% of that in the C-NC group. Conversely, in the F-HS group the total amount of insulin released in the first phase was significantly decreased to 40% of that in the F-NS group.
These findings indicate that the elevation of plasma 11-0HCS levels provoked by shocks at random rather than in a fixed time schedule is caused by the difficulty in predicting shocks, and a chronic stress induced by electric shocks at random further impairs glucose tolerance and suppresses glucose-induced insulin release in rats fed a high fat diet.

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The synergistic effects of dietary obesity produced by the feeding of a high fat diet and stress induced by electric shocks on the rates of insulin secretion, glucose utilization and oxidation in response to glucose, and the contribution of the pentose cycle pathway to glucose metabolism were examined in isolated pancreatic islets from rats fasted overnight.
Rats weighing 110 g were fed ad libitum for 12 weeks either a control (50% Starch; C) or a high fat diet (40% Butter; F). Half of the rats on each diet received electric shocks at random, 1 hour per day for the last 3 weeks of the experimental period (group C-S and F-S). The remaining rats were housed in home cages throughout the experiment (group C-NS and F-NS).
1. The rates of insulin secretion in islets from the C-NS group were increased threefold when the glucose concentration was raised from 2 to 25 mM, and the pattern of secretory response to glucose showed a sigmoid curve. The dose-response relation for glucose-stimulated insulin secretion in the C-S group was similar to that in the C-NS group. The rate of insulin secretion to glucose in islets from the F-NS group increased much more than that in the C-NS group with changes in glucose concentration between 5 to 10 mM, but the Vmax value of the secretory response was unchanged. In the F-S group, insulin secretion in islets was markedly diminished with each glucose concentration over 10 mM up to 25 mM.
2. The curves relating to the rates of glucose utilization (as judged by the formation of 3H20 from [5-³H] glucose) to glucose concentration were similar in shape to those showing insulin secretory response to glucose in the C-NS group or in the F-NS group, respectively. The rates of glucose utilization in islets from the F-S group were increased at high glucose concentration (16.7 mM) but extremely lowered at the physiological glucose concentration (from 5 to 10 mM).
3. The rate of [U-¹⁴C] glucose oxidation at 16.7 mM glucose in islets from the C groups was unchanged by stress. The rate of glucose oxidation in islets from the F-S group was decreased by 67% of that in the F-NS group or by 55% of that in the C-S group.
4. The absolute rate of glucose metabolized via the pentose cycle pathway at 2 mM glucose in islets from the F-NS group was diminished by 60% of that in the C-NS group. A marked decrease in the absolute rate of glucose metabolism via the pentose cycle pathway was observed in islets from the F-S group.
It is concluded that the decreased insulin secretory response to glucose in islets from the high fat fed rats stressed by electric shocks is associated with changes in glucose metabolism, and suggests that the decreased glucose metabolism may be involved in an inability of glucose to stimulate insulin secretion.

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In order to elucidate the process of the development and sex differentiation of the brain systems controlling gonadotropin secretion, electrophysiological studies were performed using immature intact rats of both sexes after endocrinological manipulations. Medial preoptic (MPO) unit responses to the medial amygdala (AMYG) or the dorsal hippocampus (HPC) stimulation (0.1-2.0mA, 0.2msec, 2/3Hz) and hypothalamic arcuate (ARC) unit reponses to the MPO stimulation were examined by means of post-stimulus time histograms, and percentages of neurons responding in a facilitatory or in an inhibitory manner and threshold currents for the induction of the unit response were calculated. Furthermore, the effects of gonadectomy and estrogen administration on serum gonadotropin concentrations were examined in immature female rats of various ages.
Intact rats were classified into 4 groups : i.e., 21-25, 26-30, 31-35 and 36-40 days of age. Neither responses of ARC neurons to the MPO stimulation nor thresholds of the MPO stimulation for the induction of the ARC unit responses showed any significant differences during prepuberal growth nor between sexes. In male rats, the percentages of MPO neurons which were facilitated by the AMYG stimulation decreased during the development (x²=11.963), while the change in percentages of MPO units responding to the stimulation in female rats was rather small (x²=4.053). Significant developmental changes were observed in the percentages of MPO units responding to the HPC stimulation in both males (x² = 39.957, p<0.001) and females (x²=15.628, p<0.02). Moreover, differences in the number of MPO units responding to the HPC stimulation were observed between intact 21-25-day-old male and female rats (p<0.02), between neonatally (at 2 or 3 days of age) gonadectomized 21-25-day-old males and females (p<0.005), and between neonatally gonadectomized and intact 21-25-day-old males (p<0.05), though responsive patterns of the MPO units to the AMYG stimulation were not significantly different between both sexes except at the age of 31-35 days (p<0.05).
Thresholds of the AMYG stimulation for the induction of the MPO unit responses were lowered and those of the HPC stimulation elevated during development in both sexes. Estrogen injection at weaning lowered the threshold of the AMYG, and elevated that of the HPC in female rats. Testosterone injection at weaning in male rats lowered the HPC threshold. In rats which had been gonadectomized at weaning, thresholds of the HPC stimulation were significantly lower and those of the AMYG stimulation higher than in intact rats of the same age.
Endocrinologically, a simultaneous injection of estrogen with ovariectomy caused a significant and transient increase in serum gonadotropin concentrations 1 day after the treatment in 40-day-old female rats, 2 days after in 30-day-old rats, and 3 days after in 25day-old rats.
These results showed the existence of sexual differences and developmental changes in the limbic-preoptic neural transmissions. It was suggested that the AMYG-MPO system might develop after weaning, presumably under the influence of gonadal hormones, and the lowering in thresholds of the AMYG stimulation during maturation or following estrogen treatment in female rats might be related to the development of stimulatory feedback mechanisms of estrogen which induce phasic gonadotropin release indispensable for ovulation in mature female rats. The HPC-MPO system might have a sexual dimorphism before the age of 20 days, and testes at very early periods of life as well as during postnatal growth might have an important role in the development of HPC-MPO mechanisms in the male. In female rats, estrogen might reorganize the role of the HPC, which had been a trigger for the initial FSH release for ovarian growth in the early prepuberal period,

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The factor (s) controlling the secretion of ACTH in peripheral plasma are not well known. The effects of non-extracted and extracted plasma on ACTH secretion were investigated using rat anterior pituitary cell cultures. Medium ACTH was assayed by radioimmunoassay, and the corticotropin releasing activity (CRA) was expressed as ACTH released. One hundred ul of non-extracted plasma showed significant CRA, whereas greater volumes of plasma showed reduced activity. Non-extracted plasma (250-500ul) rather reduced the secretion of ACTH evoked by hypothalamic extract (HE). When plasma was extracted with 0.2 N-acetic acid-acetone and divided into an acid phase and an acetoneether phase by adding ether, the CRA was recovered in the acid phase while no significant activity was observed in the acetone-ether phase. The acid phase extract of plasma showed a positive dose-response relationship between the amount of plasma extract (50-800 ul plasma equivalent) and ACTH release in pituitary cell cultures. The organic phase of plasma extract inhibited HE-induced release of ACTH, and this ACTH-release inhibiting activity was presumed to be corticosterone. When the acid phase extract of 20 ml plasma was applied to a Sephadex G-25 (fine) and eluted with 0.2 N acetic acid, two peaks of CRA were observed. One eluted in the region of void volume and another eluted in the retarded region where no activity was found in chromatography of HE. HE increased both ACTH and cyclic AMP release, but the plasma extract reduced cyclic AMP release. These results suggest that plasma contains both CRA and ACTH release inhibiting activity which can be extracted separately, and that plasma CRA is different from the hypothalamic corticotropin releasing factor.

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Since the discovery of the structures of somatostatin (GIF) in 1973 by Brazeau et al, its measurement by the radioimmunoassay (RIA) methods has been reported by Arimura et al (1975), Yanaihara et al (1978) and Sawano et al (1978).
As GIF does not contain tyrosine and histidine, which can be radioiodized, analogues of GIF are being used as tracers in its radioimmunoassay. In this study, two different types of trafcers (¹²⁵ I tyr⁸of GIF and ¹²⁵I-tyrosyl-GIF) were used in RIA to measure the immunoreactive GIF of fetal tissues, and their results were compared.
A highly specific anti-GIF serum was generated by immunizing rabbits with GIFconjugated BSA.
¹²⁵ I-tyr⁸-GIF and ¹²⁵I-tyrosyl-GIF were prepared by the lactoperoxidase method. The former was purified through a carboxymethylcellulose column and the latter through a Sephadex G-25 (fine) column. They were then incubated at 4°C in 0.01 M phosphate buffer saline containing 0.1% geratin and 0.2% BSA and used as tracers to measure the immunoreactive GIF-like substances in the pituitary gland, hypothalamus, pancreas, cerebrum, cerebellum, thyroid gland, stomach, small intestine and spinal cord of three fetuses obtained through legal abortion with gestation ages of 22 weeks, 23 weeks and 25 weeks. The free and bound forms were separated with the double antibody technique.
The correlation of these GIF concentrations by the two different assay systems with results such as Y=0.905X- 87.4, r=0.992 and P<0.001 was demonstrated.
From the displacement curves of the GIF-analogues (tyrosyl-GIF and tyr¹ -GIF) and the standard curves of the synthetic GIF, it was found that when ¹²⁵I-tyr⁸-GIF was used as the tracer, the anti-GIF serum could identify both the GIF-analogues and the synthetic GIF as the same substance; however, when ¹²⁵I-tyrosyl-GIF was substituted as the tracer, the anti-GIF serum could only identify the tyrosyl-GIF and the synthetic GIF as being the same substance and excluded the tyr¹ -GIF when its concentration reached beyond a certain level.
The dilution curves of the immunoreactive-GIF extracted from the hypothalamus and pancreas of the fetuses were found to be parallel to the standard curves obtained by the synthetic GIF in both asssay systems.
It is concluded that the two different assay systems which were used to measure the GIF concentrations in the fetuses can yield the same results and are useful in the application of GIF-RIA, but ¹²⁵ I-tyr ⁸ -GIF is more stable than the ¹²⁵I-tyrosyl-GIF.

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This study was performed to examine the novel mechanisms of thyroid hormone biosynthesis, by which the electron transport systems in this gland is coupled with thyroid iodide peroxidase and for this purpose, first of all, the spectrophotometric nature of the membrane-bound thyroid peroxidase was elaborated.
After a component that formed a cyanide difference spectrum at (KCN) = 5μM with a peak at 445 nm and a small trough at 410 nm had been removed by centrifugal procedure, the washed hog thyroid microsomes with sufficient peroxidase activity were used for the experiments, of which the final preparations contained 0.095 nmoles cytochrome b₅ and 0.090 nmoles oxyhemglobin per mg of proteins. The reference and sample cuvettes contained 2.5 ml of thyroid microsomal suspensions (6.7 mg of protein per ml) in 0.1 M potassium phosphate buffer, pH 7.4 and the difference spectra were measured with a Shimadzu Model 5000 spectrophotometer and Union Giken Model SM 401 spectrophotometer at 25°. Since the peroxidase in the thyroid microsomes was specifically inactivated by NADPH under aerobic experimental conditions, it became possible to obtain spectral data on the enzyme bound to the microsomes. And the spectral characteristics of hog thyroid peroxidase bound to microsomes were compared with those of the peroxidase-inactivated microsomes, solubilized thyroid peroxidase and some other purified hemoproteins.
Spectrophotometric titrations of hog thyroid microsomes with cyanide and azide gave monophasic dissociation curves having dissociation constants of 17 and 27 μM, respectively, at pH 7.4. These values were approximately equal to the concentrations of cyanide and azide that caused half-maximal inhibition to the peroxidase activity of the microsomes. Upon the addition of NADPH to an aerobic suspension of the microsomes, the Soret bands of oxidized and reduced thyroid peroxidase decreased with a concomitant decrease in the peroxidase activity. From the difference spectra between the intact and peroxidase-inactivated microsomes, the Soret band of the enzyme was assumed to be at 410 nm in the ferric form and at 421 nm in the “stable” ferrous form. Though it has so far been generally regarded that thyroid peroxidase is similar to horseradish peroxidase, the following proterties of the membrane-bound thyroid peroxidase obtained from the present study were markedly different from those of hemoglobin, myoglobin and horseradish peroxidase, but rather similar to those of lactoperoxidase. The affinity of the reduced enzyme for cyanide was relatively high and a cyanide difference spectrum was observed in the CO saturated suspension of reduced thyroid microsomes. This difference spectrum which represents cyanide complex of the reduced form of thyroid peroxidase in sample cuvette and CO complex of the reduced form in reference cuvette can be the most suitable and characteristic one to detect the spectrum of thyroid peroxidase present in thyroid microsomes, especially for clinical materials. Because the amounts of thyroid microsomes prepared from patients are usually extremely limited and even if methemoglobin is present or generated through autoxidation in thyroid microsomes, for example, in the case of aging and so forth, this difference spectrum can not be affected by the presence of methemoglobin. Furthermore, the reduced enzyme as well as the oxidized enzyme forms a spectrophotometrically detectable complex with azide at neutral pH under aerobic condition in the presence of CO, differently from horseradish peroxidase.
From these data in addition to the labile property of thyroid peroxidase to H₂O₂, it appears that this enzyme is an anomalous type of peroxidase compared with the other known enzymes. The nature of thyroid peroxidase prepared by non-proteolytic method,

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Enzyme-linked sandwich immunoassays for the measurement of thyroglobulin and anti-thyroglobulin autoantibody in human serum using silicone rod and β-D-galactosidase were studied. These methods showed excellent results in specificity, sensitivity, precision and clinical application.
1) A method using silicone rod coated with rabbit (anti-human thyroglobulin) immunoglobulin G and rabbit (anti-human thyroglobulin) monovalent fragment of immunoglobulin G (Fab') conjugated with β-D-galactosidase was developed for the measurement of circulating thyroglobulin. The sensitivity of the assay with as little as 2 μl of serum was 10.7 amoles/tube corresponding to 3.5 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The correlation coefficient between values determined by the present assay and a double-antibody radioimmunoassay was 0.99 (n = 63, p<0.001). Circulating thyroglobulin was detectable in 90% of 146 normal subjects, the concentration being 13.3 ± 10.3 ng/ml (mean ± S.D.). Interference of anti-thyroglobulin autoantibody with the assay for thyroglobulin was smaller that' that in radioimmunoassay.
2) Another method using human thyroglobulin conjugated with 0-D-galactosidase and silicone rod coated with human thyroglobulin was developed for the measurement of circulating (anti-human thyroglobulin) autoantibody. The sensitivity of the assay with as little as 5 μl of serum was 7 fmoles/tube corresponding to 220 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The highly significant correlation was observed between the concentrations of anti-thyroglobulin autoantibody determined by the present assay and a radioimmunoassay (r = 0.80, n = 74, p<0.001) and also between those by the present assay and those by tanned red cell hemagglutination (r = 0.78, n = 119, p<0.001). No effect of thyroglobulin on the present assay was observed unless the ratio of the amount of thyroglobulin to that of (anti-human thyroglobulin) immunoglobulin G was higher than a tenth.

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Thyroid peroxidase (TPO) was partially purified from hog thyroid microsomes after solubilization by means of deoxycholate treatment followed by ammonium sulfate fractionation and affinity chromatography with Con A Sepharose. The absorption spectra of the preparation showed the maxima at around 410 nm for oxidized form, 422 nm for dithionitereduced form and 422 nm for CO complex of reduced form. The cyanide difference spectrum showed a peak at 431 nm and a trough at 403 nm. This preparation was contaminated with little cytochrome b5 and it was shown that the TPO preparation was able to be used for the following spectrophotometric experiments.
The addition of H₂O₂ to the TPO preparation induced the characteristic change in the difference spectrum with a peak at 430 nm and a trough at 407 nm, which was gradually disappeared in a few minutes, but at the high concentration of H₂O₂ (35 μM) the trough at 411 nm was observed after decomposition of H₂O₂ accompanying loss of peroxidase activity. This deepening of the trough caused the heme degradation which was dependent with the concentration of H202 added and to less extent at the low concentration of H202 (3.5 μM). Since the difference spectrum produced by the addition of small amount of H₂O₂ disappeared rapidly after the addition of KI or ascorbate and resembled the spectral change due to the formation of Compound II in the reaction of other perioxidases, it was concluded that the difference spectrum with a peak at 430 nm and a trough at 407 nm observed after the addition of H₂O₂ was ascribable to the formation of Compound II of TPO. Although Compound I was not observed under the experimental conditions used, the results were accounted for the presence of I bound to TPO or other endogeneous reducing agents. We tentatively concluded that Compound I and Compound II are formed in the reaction of TPO with H₂O₂ as well as in that of horseradish peroxidase.

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