Marked discrepancies have previously been observed between insulin-like activities by rat fat pad assay (ILA) and insulin levels by radio-immunoassay (IRI) for the native sera. As miscellaneous factors in the serum may affect these assays, separation of serum insulin by means of salt-ethanol extraction, acid-ethanol extraction or gel filtration was carried out prior to assay. Samples were dog sera obtained from the pancreatic and femoral veins, either at fasting or after glucose injection. The salt-ethanol procedure originally devised by Romans, was modified for the extraction of serum insulin. Recoveries of endogenous IRI by the salt-ethanol and the acidethanol procedures were 90% and 72% respectively, almost identical with recoveries of 131I-insulin added to sera. By gel filtration with Sephadex G 50, 131I-insulin was eluted separately from most of serum proteins. Serum was separated by this method into two fractions, one of which (Fr. A) contained most of serum proteins, and the other (Fr. B) was eluted following Fr. A corresponding to the peak of 131I-insulin. In contrast to discrepancies of ILA and IRI in the native sera, ILA and IRI values of these extracts and fraction B were much closer to each other. These data demonstrate that IRI behaves like the pancreatic insulin in extraction and gel filtration procedures, and suggest that serum endogenous insulin assayed as IRI also possesses biological activity corresponding to its immunological reactivity. Fraction A by gel filtration, despite the lack of any appreciable IRI, showed a marked ILA by fat pad assay. The ILA of a depancreatized dog was measured as 405μU/ml, although its IRI was undetectable. This ILA was not recovered by either extraction procedures nor in Fr. B by gel flit ration, but a maked ILA was detected in Fr. A. Fraction A, as well as insulin and fraction B, stimulated various parameters of fat pad metabolism such as glucose uptake, net gas exchange, 14C incorporation from glucose-U-14C into CO2, total lipid and glycogen. Cysteine treatment suppressed ILA of both Fr. A and Fr. B, while anti-insulin serum suppressed ILA of Fr. B but not that of Fr. A. Four point assay by fat pad revealed parallelism between the extracts or fraction B and crystalline insulin, but deviation from parallelism was significant between Fr. A and insulin. These results suggest that the difference between ILA and IRI is mainly attributable to the ILA in fraction A. This ILA is probably due to factors entirely different from crystalline insulin, although it possesses many similar biological effects with insulin on adipose tissue metabolism in vitro.
We studied the oxidative phosphorylation and the respiratory control of liver mitochondria in normal and alloxan diabetic rats. We obtained the following results. 1) Respiratory control index and ADP/O for alloxan diabetic liver mitochondria were found to be lower than that of control. 2) Low levels of respiratory control index and ADP/O for alloxan diabetic rats recovered to normal after injection of insulin, but did not rise by addition of insulin in vitro. 3) Insulin blocked the respiratory release by D. N. P. in mitochondria suspension of rats liver.
Male albino rats were devided into three groups and fed respectively on three kinds of laboratory fodders;standard diet, high sucrose diet (50% sucrose in caloric) and high fat diet (50% lard in caloric). Following the feeding on these special diets for 8-28 days, 50 mg per kg body weight of alloxan was injected subcutaneously four times, and 75 mg per kg body weight of alloxan twice every week or two weeks. The rats in these three groups were fed on isocaloric diet through all period of this experiment. 1) Diabetes was not found in the standard diet group (9 rats). 2) Seven of the high fat diet group (12 rats) became diabetic, and ketonuria was proved in three of these seven diabetic rats. 3) Three of the high sucrose diet group (13 rats) became diabetic, ketonuria was not found in these three rats. Although there was no diabetic rat in the standard diet group, severe diabetes was found in 58% of rats in the high fat diet group. Therefore it was considered that the diabetogenic effect of alloxan was intensified with the high fat diat.
During the year 1964, 128 diabetics were hospitalized in this institution. They were treated separately with insulin and SU, and the responses shown by them were closely observed, and it is possible to arrange them into the following 6 classes according to the characteristic responses to these substances. I. Classification A-group: excellently effective with insulin as well as SU, 43%. B-group: refractory to insulin or SU alone, 14%. C-group: so-called insulin dependent diabetics, non-responsive to SU, 14%. D-group: pseudo-SU-resistant diabetics, good response to SU after repeated longterm insulin therapy, 5%. E-group: poor response both to insulin and SU, 8%. F -group: well-controlled under dietary restriction alone, 16%. II. Clinical Characteristics (a) The A-group which showed the best response to insulin as well as SU, gave the most favourable results on the GTT test done before and after the treatment; but the C- and D-groups were found to be the worst. (b) The insulin sensitive test (judged by the variation of blood sugar after insulin subcutaneous injection) revealed in the B- and E-groups many insulin resistant patients meaning the usual insulin action was inhibited, and many others showed the insulin high rebound effect. (c) With regard to the tolbutamide sensitive test (similar to (b) after intravenous injection of tolbutamide), no change whatever of blood sugar was seen in the C-group all through the treatments; the D-group responded at the commencement of the treatments with no lowering, but near the end of the treatments showed the lowering effect while in the others there occurred invariably the lowerings when no hepatic or retinopathic complex was accompanied. (d) Accompanying diseases differed from one group to another: thus (1) retinopathy was found common in the C- and D-groupes, but quite less in the B- and E-groups; and (2) hypercholesteremia showed any marked differences in all the groups. Under restriction of fat diet, the relation hypercholesteremia and insulin therapy was observed, and found the high level blood cholesterol deceased in fairly many cases in the C- and D- groups.
The maximum conduction velocities of the sciatic nerve of diabetic dogs were measured in vitro. Diabetic state was produced by intravenous injection of alloxan. The nerve conduction velocities of 4 diabetic dogs measured on 7 days after the alloxan injection were in normal range, and the velocities of other 4 diabetic dogs on 10 days after the alloxan injection were also in normal range. However the conduction velocities of 8 diabetic dogs measured on 14 days after the alloxan injection decreased satisfactorilly significant (p<0.01), and there was no relationship between rates of the decreses of conduction velocities and blood sugar levels on 14 days after the injection.
There have been few follow-up studies on diabetes for a long period in Japan. The results of the present study were as follows: 1) Only 319 patients among 725 diabetics treated in our clinic in a period from 1956 to 1961 could be subjected for the follow-up study over 5 years, because remaining 406 cases had lost the contact with our clinic or died within 5 years. 2) In 1966, only 120 subjects among 319 diabetics followed-up were visisting regularly our clinic. 3) About 40% cases of the total 319 diabetics in the follow-up study had been treated with insulin over 5 years. Cases using insulin among the follow-up cases seemed more frequent than those among patients who failed to be followed-up. 4) Forty-three diabetics among 319 cases died after the follow-up over 5 years. Main causes of the death were cerebro-vascular diseases and malignant tumors in the male diabetics and nephropathies in the female diabetics in our cases.
A Japanese female, 53 years of age, has noticed multiple non-itching, painless, small erythematous lesions on both legs in 1960. The lesions gradually increased in size and their surfaces became brown and atrophic in succeeding several months and remained unchanged until her administration in May, 1964. She has been diagnosed as Grave's disease in 1953 and diabetes mellitus in 1956. On admission, pulse rate was 80/min. and blood pressure was 150/52 mmHg. Medium sized goiter was noticed. Lung, heart and abdominal organs were normal. Glucose tolerance test revealed mild diabetes mellitus. BMR was +26%, PBI 5.6γ/dl, 1311 uptake 69%, serum total cholesterol 228 mg/dl, and aminoacid nitrogen 8.8 mg/dl. Serum elect-rolytes and liver function tests were normal except for considerably high alkaline phosphatase level. Biopsy specimens showed necrobiotic changes of collagen fibers, i.e. degeneration of fibers, which consisted of epitheloid cells, histiocytes and giantcells of foreign-body type, and perivascular inflammatory reaction. Some small fat droplets which were stained reddish brown by Sudan were found extra- and intra-cellulary. Macroscopic and microscopic skin changes coincided with those of necrobiosis lipoidica diabeticorum. Although more than 300 cases have been reported in the literature since first description of Oppenheim in 1929, this disease is said to be rare in non-white races. Only one reliable case has been reported in Japan by Matsubara and Hori in 1961. Other reported cases are uncertain because of their unsatisfactory description.