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A modified erythrocyte insulin receptor assay was developed. The major advantage of our method is the smaller amount of blood necessary for the assay and consequent simple handling in the binding assay. Three ml of blood is enough for this assay, making it possible to assess insulin receptors in a group of pediatric patients. There was a close relationship between mononuclear cells and erythrocyte insulin binding assays (r = 0.900, p0.05). With this method insulin binding studies were done on normal subjects and adult onset non-obese diabetics. Decreased insulin binding was seen in the diabetic group, and this was due to a decreased number of insulin receptors. This erythrocyte insulin receptor assay can be useful tool for the study of the mechanism of insulin resistance.

We studied the in vivo and in vitro effect of p-chlorophenoxyisobutyrate (CPIB) on insulin binding and glucose transport in isolated rat adipocytes. In the in vitro study, adipocytes were incubated with 1mM of CPIB for 2 h at 37 degrees C, pH 7.4, and then insulin binding (37 degrees C, 60 min) and 3-0-methylglucose transport (37 degrees C, 2s) were measured. Incubation with CPIB did not affect either insulin binding or glucose transport in the cells. The addition of insulin (10 ng/ml) with CPIB to the incubation media also did not affect the following insulin binding and glucose transport. In the in vivo study, rats were fed a high sucrose-diet containing 0.25% CPIB for 7 days. Serum cholesterol, plasma free fatty acid, and insulin levels were significantly decreased in the CPIB-treated rats. The treated rats demonstrated an almost 2 fold increased maximal binding capacity for insulin (189,000 sites/cell for treated vs 123,000 sites/cell for control cells). Basal glucose transport (glucose transport in the absence of insulin) significantly decreased in the CPIB-treated rats, although insulin-stimulated glucose transport was comparable in treated and control cells. Thus, CPIB might have no direct effect on glucose transport and insulin binding, as determined by the in vitro studies. Furthermore, a relatively short-term in vivo treatment with CPIB, such as 7 days, did not stimulate glucose transport.

We studied insulin degradation in human cultured lymphocytes (RPMI-1788 line) with a small but significant number of lysosomes under the electron microscope. Insulin degradation determined by the TCA solubility method was 64.6 +/- 1.2% (mean +/- SEM) at a trace concentration after the incubation with 2.0 x 10(7) cells (4.0 x 10(7) cells/ml) for 60 min at 37 degrees C. Because insulin degradation was 54.6 +/- 7.0% in the cell-free buffer in which 2.0 x 10(7) cells were previously incubated, most of the insulin was degraded outside of the cells. Gel filtration of the radioactive materials also revealed that most of the labeled insulin in the medium was degraded, and the main peak of the cell-associated radioactivities was intact labeled insulin. Chloroquine, a lysosomotropic agent, failed not only to increase insulin binding but also to decrease the insulin degradation. Other lysosomal protease inhibitors, antipain and leupeptin had also no effect on insulin degradation. In contrast, bacitracin (500 micrograms/ml) significantly decreased the insulin degradation analyzed by TCA solubility, receptor-rebinding, and the gel filtration method. These results suggest that insulin molecules are degraded by the enzymes leaked from the cells. The non-receptor mediated process, which is the bacitracin sensitive pathway, might be a general mechanism of insulin degradation in human cultured lymphocytes in vitro.

We compared A-14 and A-19 125I-labelled insulin in receptor-binding and degradation. Percent receptor-binding of A-14 and A-19 125I-labelled insulin to 2.4 X 10(9)/ml erythrocytes after 210 min incubation at 15 degrees C was 7.8 and 4.9%, respectively. Percent insulin-receptor binding of A-14 insulin was 1.6 times greater than that of A-19 insulin. A similar result was obtained in an adipocytes insulin binding study. Percent receptor-binding of A-14 and A-19 insulin to 2 X 10(5)/ml fat cells after 30 min incubation in the above buffer was 3.9 and 2.4%, respectively. Degradation of A-14 and A-19 insulin in rat adipocytes was also studied by molecular sieve column chromatography. Isolated rat adipocytes were allowed to associate with A-14 and A-19 125I-insulin for 60 min at 37 degrees C, pH 8.0 in a HEPES-phosphate buffer, and then cells were separated from the buffer by centrifugation. After solubilization with triton X-100, both the solubilized cells and the incubation medium were applied to the Bio-Gel P-30 column to assess the insulin degradation. Degradation of A-14 125I-insulin by the isolated rat adipocytes was 1.6 times greater than that of A-19 125I-insulin. Furthermore, the peak which was thought to be intermediate degradation products of insulin was obtained between the peak of intact insulin and that of 125I-tyrosine. Such a peak of intermediates was much smaller in the incubation media than in the cell-associated materials.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloxan diabetic rats with fasting blood glucose levels above 300mg/100ml were treated with oral administration of water-in-oil-in-water (W/O/W) insulin emulsions at a dose of 50 U/100g body weight, three times daily for 10 to 14 days. The course of diabetes was followed by determinations of glucose levels in blood and urine.During treatment with oral W/O/W insulin emulsions, daily excretion of urinary glucose decreased by about 30 to 40%(2 to 3g/day) in all of the five rats studied, and returned to the pre-treatment levels after the treatment being discontinued. During treatment, a significant reduction in fasting blood glucose levels was observed in 4 out of 5 rats, giving the decrease by 18 to 44%. Quantitative estimates suggested that the effectiveness of 50 U/100g of oral W/O/W insulin emulsions was comparable to that after intramuscular regular insulin at a dose of 0.5 U/100g.Although oral W/O/W insulin emulsions are still of low efficiency, these results would indicate that diabetes can be controlled by effective oral insulin preparations.

C14-incorporation into blood glucose and liver glycogen from C14-labelled alanine was studied in rats with acute or chronic CCl4 liver damage.In rats with CCl4 liver damage, C14-incorporation from alanine into blood glucose and liver glycogen was found to be decreased, especially in rats with acute CCl4 liver damage. Such a disturbed C14-incorporation was improved by the administration of 2.5mg of cortisone to rats with chronic CCl4 poisoning. However, in rats with acute CCl4 liver damage, cortisone treatment caused a marked increase in C14-incorporation into blood glucose and failed to increase C14-incorporation into liver glycogen.The latter finding may be related to the increased incidence of steroid diabetes in patients with liver damage.

Using an everted sac technique, the intestinal absorption of insulin in the rabbit has been investigated.Insulin was shown to cross the intestinal wall, as demonstrated by the immunoreactive insulin inside the sacs (serosal fluid). The rate of insulin transference for a period of one hr was small, corresponding to approximately 0.1% of the insulin added to the mucosal fluid. This rate was not inhibited by the addition of 2, 4-dinitrophenol.The small transference rate would indicate that the submucosal and muscular tissues of the intestinal wall, in the absence of vascular perfusion, present a barrier to the movement of large molecular weight substances.

We studied the effect of acute short-term exercise on insulin binding to erythrocytes in patients with non-insulin dependent diabetes mellitus and normal controls. In the normal controls, insulin binding was increased immediately after the exercise and returned to the basal level at 30 min after the exercise. This increase was due to an affinity change without any change in the insulin receptor number. In contrast, insulin binding tended to decrease in the diabetics after the exercise, and this change was due to a decreased affinity without any change in the number of insulin receptors. These results indicate that the control mechanism of insulin receptor affinity in response to exercise is different in non-insulin dependent diabetics and normals.

The effect of propranolol treatment (60 mg per day, three days) on glucose disposal rate (K-value) was investigated in nine patients with hyperthyroidism. K-value was improved in 4 cases and aggravated in 5 cases. 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.

Insulin binding to erythrocytes from normal subjects over a wide range of ages (group I, cord blood; group II, Age 6M to 6 yrs; group III, 7 to 19 yrs; group IV, 20 to 59 yrs, group V, over 60 yrs of age) was determined using modified Gambhir's method and the effect of age and sex on the binding was evaluated. The highest insulin binding to cord blood erythrocytes was accounted for by the increased number of reticulocytes since there was a highly significant correlation between insulin binding and the reticulocyte count (r = 0.928, p less than 0.001). Group IV showed significantly higher binding than younger are groups II and III. The mean binding in group V was the highest among all age groups except for group I, although the differences failed to reach statistical significance. The receptor concentrations (R0) were comparable among groups except group I which had a slightly increased R0. The affinity of insulin receptors was the lowest in group I and II, and gradually increased with the age and a positive correlation (p less than 0.01) was obtained between age and Ke. This increase in affinity with age is considered an adaptive response to the intracellular metabolic derangement often seen in the aged. No effect of sex on insulin binding was seen before puberty. However, in normal adults insulin binding of the female tended to be lower than that of the male with slightly increased R0 and decreased affinity in the female. Thus, the slightly decreased binding of the female group was mainly accounted for by the decreased affinity although the difference in the affinity was not significant.

In order to determine whether glucagon has a ketogenic effect through the activation of hepatic ketogenesis or not, 1mg of glucagon was injected intravenously in bolus in normal and in mild or severe (insulin-dependent) diabetics, and plasma levels of 3-hydroxybutyrate (3-OHBA) and free fatty acid (FFA) were measured.The ketogenic effect of insulin-free glucagon was also tested in the diabetic rats. The effect of (+)-decanoylcarnitine, an inhibitor of hepatic fatty acid oxidation at a step catalyzed by carnitine palmitoyltransferase, upon the ketogenic action of glucagon was also studied in the diabetic rats.In normal and in mild diabetics, glucagon had no effect on plasma 3-OHBA concentration, but a significant elevation (0.55m mole/l at 30min) was observed following glucagon in insulin-dependent diabetics. The hyperketonemic effect of glucagon was most marked (1.6m mole/l at 30 min) in those who exhibited mild ketonemia before the glucagon injection. No comparable rise of plasma FFA was observed in these subjects. In normal subjects, plasma FFA significantly decreased following glucagon, presumably due to the anti-lipolytic effect of released endogenous insulin.In normal rats, insulin-free glucagon (0.5mg iv) produced a small but significant rise of plasma levels of FFA and 3-OHBA within 20 min. In diabetic rats, the greater rise of plasma 3-OHBA (1.4m mole/l at 20 min) following glucagon was observed without any rise of FFA. And this rise was abolished by the concomitant injection of (+)-decanoylcarnitine.These results indicate that glucagon has ketogenic action through the activation of hepatic ketogenesis under conditions where insulinopenia and the absence of endogenous release of insulin coexist.

Of 9 patients with chronic hepatitis treated with intravenous administration of 40 to 200 mg/day of glycyrrhizin, 3 diabetic patients receiving concomitant insulin developed hypokalemia, sodium retention and suppression of both plasma aldosterone concentration and plasma renin activity after the administration for 3 to 6 days. In the remaining 6 patients (5 nondiabetic and 1 diabetic) receiving no insulin, the administration over the long term (18 to 266 days) never caused these abnormalities. The development of hypokalemia and sodium retention in the patients was not associated with increased urinary excretion of potassium, indicating a different condition from pseudoaldosteronism caused by the desoxycorticosterone-like action of glycyrrhizin. These findings suggest that insulin which is known to have hypokalemic, antinatriuretic and antikaliuretic activity, as well as glycyrrhizin plays an important pathogenetic role in the observed electrolyte disturbance, and suppression of both renin and aldosterone.

Clinical experience with long-term sulfonylurea treatment has been discussed in the management of diabetes mellitus. Because of the close relationship between fasting serum insulin-like activity and the rate constant for glucose utilization (K) after glucose loading, the latter values were used for screening diabetics in place of the serum insulin-like activity, except for severe diabetics with K value below 0.04. Oral anti-diabetic drugs, such as sulfonylurea, were not effective in diabetics who had no significant extra-insulin secretion reserve. Secondary failure was found to occur more rapidly and more frequently in diabetics with low fasting serum insulin-like activity. In diabetics with low fasting serum insulin-like activity but with relatively high extra-insulin secretion, these drugs were significantly effective and secondary failure developed less rapidly.