Using human skin fibroblasts, adapted in cell culture, age-related changes in cellular responsiveness to insulin and binding of ¹²⁵I-insulin to the cells were studied.
The mean age of the skin donors was 19 yr in the case of the young group of cells (N=4) and 75 yr in the aged group (N=5).
Experiments using the cells in the growth phase, in which the cellular responsiveness to insulin was more marked than in the phase of confluent monolayer, yielded the following results.
1) The insulin stimulated glucose uptake was significantly smaller in the cells from aged donors than in those from young donors.
2) The conversion of glucose to CO₂ was also significantly smaller in the cells from aged donors than in those from young donors.
3) The maximal specific binding of ¹²⁵ I-insulin to the cells from aged donors was significantly smaller than that to the cells from young donors.
4) No significant difference in the effect of insulin on the binding of ¹²⁵ I-insulin was observed between the young and aged groups of cells.
5) Also, no significant differences between the two groups of cells were observed in the effects of time, temperature and pH on the binding of ¹²⁵I-insulin to the cells.
These results suggest that a decrease in the number of binding sites of insulin occurs with advancing age without qualitative changes, and that this age-related change in binding sites of insulin represents one of the possible mechanisms for the age-related reduction in cellular responsiveness to insulin.

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The incidence of diabetes mellitus is greatly influenced by environmental factors such as food intake, physical activity, emotional stress, etc. Many reports have shown that the prevalence of diabetes has increased remarkably during the last 20 yr in Japan. This led us to determine whether the overall blood sugar level of the Japanese is on the increase or not. To clarify this problem, measurements of fasting blood sugar were made in employees Lof small-or medium-sized factories and firms located in Tokyo, who visited our station (Center) for annual health examinations during the period 1969-1977 (5, 000-40, 000 persons yr). The blood sugar was estimated by Hoffman's method using an autoanalyzer. In general, the mean blood sugar values for the years 1975-77 were higher than those for 1969-70, and this tendency was more marked in males thanfemales and in older rather than younger people. The differences in mean fasting blood sugar values between 1969 and 1977 were 3.7 mg/100 ml, 3.9 mg/100 ml and 3.8 mg/100 ml for males in their 30's, 40's and 50's, respectively. The corresponding figures for females were 1.0, 2.6, and 2.4 mg/100 ml, respectively. The frequency distribution curve for fasting blood sugar shifted to the right with advance of calendar year. The causes of this phenomenon are thought to be the changes in dietary habits and rapid changes in socioeconomic conditions that have occurred recently in Japan.

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It is well known that the abnormalities of the glucose tolerance test are often found in patien with Graves' disease. Moreover, the coexistence of Graves' disease, which is regarded as a autoimmune disease, with diabetes mellitus has been noted by several workers. Even so, tl characteristics of overt diabetes mellitus in Graves' disease have not yet been reported in detail.
The occurrence of overt diabetes mellitus was therefore studied in 270 patients with Graves' disease, and the characteristics of the patients were investigated. Seven patients (2.6%) with overt diabetes were found among the 270 patients with Graves' disease. They were all women and most were over 45 yr old. They were characterized by having mainly insulin-dependent diabetes and by showing extremely high titers of anti-microsomal antibody. The family history of 4 patients revealed that one or more close relatives had diabetes mellitus. Moreover, histological findings for specimens obtained by thyroid needle biopsy in 3 of the 7 cases revealed the coexistence of histological features of Hashimoto thyroiditis with the proliferative changes of epithelial cells characterizing Graves' disease, indicating hashitoxicosis histologically. Furthermore, none of the patients suffering from Graves' disease with associated overt diabetes, displayed complications of diabetic retinopathy or evidence of albuminuria.
It has previously been reported that prolonged administration of thyroid hormones can cause the overt diabetes mellitus in animals which is known as metathyroid diabetes. In the present study, one patient with a diabetic tendency in her family showed an apparently diabetic pattern in her glucose tolerance curve and a markedly low response of IRI about 10 months after the onset of clinical signs and symptoms of Graves' disease indicating human metathyroid diabetes. On the other hand, the low incidence of overt diabetes in patients with Graves' disease suggests that the diabetogenic action of thyroid hormones may not necessarily be the important factor in causing the diabetes mellitus. Thus, the role of other factors including abnormalities of the immune mechanism must be considered in relation to the coexistence of Graves' disease and diabetes mellitus.

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The correlation between diabetic retinopathy and coagulation factor XIII was investigated by analyzing the plasma enzyme in patients with each grade of retinopathy.
The subjects consisted of 35 diabetics with retinopathy of Scott degree I-V, 23 diabetics without retinopathy, and 62 controls.
Plasma factor XIII was determined by the fluorescent method.
The results revealed no significant differences in average factor XIII level between non-diabetics and diabetics with or without retinopathy, but did indicate several facts as follows.
1) The plasma factor XIII concentration is high in 65 to 80-yr-old patients with any grade of retinopathy and in 35-to 64-yr-old with retinopathy of Scott degree II.
2) The deviation in plasma factor XIII concentration increases in diabetics with retinopathy in excess of Scott degree II, and also, the value of factor XIII increases in subjects with a history of poor control of blood sugar.
3) Several determinations of factor XIII in each of the cases with diabetes mellitus revealed a simultaneous increase in standard deviation rate of factor XIII and fasting blood sugar (r=+0.725).
Abnormal deviations in plasma factor XIII level in the course of the disease may possibly be due to consumption and regulatory supply often being in excess, assuming that proliferative retinopathy is based on chronic localized intravascular coagulation.

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We have developed an artificial beta cell, which is a closed-loop control system elaborating measurement, communication and operation automatically, to normalize the blood glucose concentration of diabetics on a minute-by-minute basis. To establish the insulin infusion program, adaptive control theory was applied. First, the dynamic property of glucose-induced insulin secretion was simulated with control theory and the relationship between glucose concentration and insulin secretion was expressed in the transfer function of proportional (glucose concentration per se) and derivative (rate of change in glucose concentration) action to glucose concentration.
Then, utilizing this model, the following computer algorithm was prepared:
I. I. R.=KpBG+KdΔBG+Kc,
where I. I. R. is the rate of insulin infusion, BG is blood glucose concentration, ΔBG is the rate of change in glucose concentration per min, and Kp, Kd and Kc represent the coefficient for proportional action, the coefficient for derivative action and the constant for basal insulin secretion, respectively.
The artificial beta cell system consisted of 4 subunits, as follows: 1) continuous glucose measurement with a Technicon Autoanalyzer II using modified manner from the glucose oxidase method and the time delay between blood withdrawal and the monitoring of blood glucose amounts to 4 min, 2) a microcomputer system forecasting the blood glucose concentration at 4 min ahead and calculating the insulin infusion rate every minute, 3) an insulin infusion mini-pump, and 4) a printer resistering all output records. Subunits 2), 3) and 4) were all kept in a small case for bed-side use. Body weight and other parameters were fed into the microcomputer system arbitrarily according to the condition and insulin secretory ability of the individual subject.
Perfect normalization of blood glucose response following test-meal intake and therapy of the hyperglycemic-ketotic state in diabetic patients were successfully attained with this system.
The characteristics of the system as recognized in the clinical applications were: 1) the rate of insulin infusion was sufficiently small to simulate plasma concentrations of insulin exactly the same as those seen in normal subjects, 2) insulin requirements were so reduced to around half of those given subcutaneously, and 3) glucose or glucagon was not essential to rectify hypoglycemia.
This artificial beta cell system is considered useful not only for clinical applications, but also as an elegant research tool for further investigations of carbohydrate, lipid and amino acid metabolism and the action of anti-insulin hormones.

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Tolbutamide has been reported to be an uncoupler of oxidative phosphorylation of liver mitochondria. However, its mechanism of uncoupling is not fully elucidated. We obtained the following results by measuring oxygen uptake and the swelling of rat liver mitochondria in the presence of tolbutamide and Ca²⁺.
1) In the presence of O.1 mM EGTA, respiration of liver mitochondria was not influenced by the addition of 1 mM tolbutamide or 0.3 mM Ca²⁺. However, controlled respiration of mitochondria was released by the concomitant addition of 1 mM tolbutamide and 0.3 mM Ca²⁺.
2) In the presence of 0.1 mM EGTA, the swelling of liver mitochondria was not influenced by the addition of 2 mM tolbutamide or 0.3 mM Ca²⁺. However, the concomitant addition of tolbutamide and Ca²⁺ induced drastic swelling of liver mitochondria.
3) Addition of O.2 mM EGTA caused partial restoration of the uncoupling action of 3.8 mM tolbutamide. Addition of 0.2 mM EGTA and O.4 mM Ca²⁺ accelerated the uncoupling action of 2.0 mM and 3.8 mM tolbutamide.
The above results indicate that tolbutamide changes the ability of the membrane to allow Ca²⁺, either outside or inside the mitochondria, through the membrane.

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Although radioimmunoassay (RIA) of insulin has greatly contributed to basic and clinical medicine, its utilization is limited only to certain laboratories with specific facilities and qualifications for using the radioisotopes. A method which does not require radioisotopes but is capable of detecting microquantities of the peptide hormone in the blood has thus been keenly sought. The present EIA method employs a sandwich technique based on the formation of a complex: insulin antibody attached to a plastic bead-insulin of the serum sample-insulin antibody labeled with horseradish peroxidase. The amount of complex formed is proportional to the amount of insulin in the sample.
The method was found to be as sensitive and reproducible as RIA. Standard curves for EIA were linear in the range of 0-160 μU/ml of insulin. The minimum detectable dose appeared to be 5 μU/ml. The recovery rate of exogenously added human insulin in a given sample was 95% on average. Dilution of a sample in the ratio 1: 2 to 1: 8 by a known insulin concentration of human serum gave satisfactory results. The coefficients of intra-and interassay variation were found to be 5% and 9%, respectively. Serum insulin levels during oral glucose tolerance tests as determined by both EIA (Y) and RIA (X) based on a single antibody method, were parallel at all sampling times. The regression line and correlation coefficient between X and Y were: Y (μU/ml) =7.67+1.22 X (7MU;U/ml), and r=0.803 (p<0.001). When RIA was determined by a single antibody method (X₂), the relations between X₂ and Y were: Y (μU/ml) =0.24+1.01 X₂, and r=0.952 (p<0.001).
The essentials for carrying out this EIA method properly can be summarized as follows. 1) Serum samples, if available, are preferable to plasma samples. 2) A serum sample with a known concentration of insulin is necessary for the dilution of the given serum sample, if required. 3) The assay tube must contain a certain amount of either rabbit or human serum to obtain a reasonable EIA value.

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We have reported the successful clinical applications of a bedside-type artificial beta cell system in diabetics. However, the ultimate goal of the system is to control the patients' blood glucose over long periods of time. In view of the difficulties in developing a of portable glucose sensor, a small and light pre-programmable insulin infusion pump was devised for clinical use. The time pattern of insulin infusion rate for 24 hr obtained with the aid of an artificial beta cell was pre-programmed in to in the computer, which controlled the pump. The general insulin infusion regimen was as follows: the rate of intravenous insulin infusion for 2 to 3 hr after each meal was about 5 to 8 times B (B=225 μU/kg · min), and the constant rate B was maintained for the rest period.
It was found that small amounts of intravenous insulin produced adequate glucose homeostasis, far superior to the states induced by much larger doses of subcutaneous insulin. The quality of control was consistent in all the cases studied.
Thus, as an alternative to the artificial beta cell alone, a combination of the artificial beta cell with a pre-programmable insulin infusion pump was shown to be capable of restoring the circadian blood glucose profiles of diabetics to within the physiological range. This combination is thought to be useful for the long-term treatment of ambulant diabetic patients.

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