Leak of lysosomal enzymes is a possible indicator of permeability of the cell membrane. Acceleration of such permeability in diabetics may cause diabetic microangiopathy. Elastase is one of the lysosomal and collagen-hydrolytic enzymes. The activity of elastase in plasma and granulocytes of diabetics was measured by the method of Katagiri using succinyl-trialanine-p-nitroanilide. Results are presented as the mean±SD.
The enzyme activity in the plasma of diabetics (0.073±0.018/h) was significantly (p<0.001) greater than that in the plasma of non-diabetics (0.062±0.012/h). The enzyme activity in the plasma of diabetics was 0.068±0.020/h for the diet group, 0.071±0.014/h for the group treated with oral hypoglycemic agents and 0.080±0.016/h for the insulin group. There were significant differences between the enzyme activity of plasma in non-diabetics and that in plasma of the group treated with oral hypoglycemic agents (p<0.05), and between the enzyme activity in plasma of non-diabetics and that in plasma of the insulin group (p<0.001).
The enzyme activity in the plasma of diabetics with proteinuria (0.082±0.015/h) was significantly (p<0.01) greater than that in the plasma of diabetics without proteinuria (0.068±0.017/h). There was no significant difference between the enzyme activity in the plasma of diabetics with and without retinopathy. There was no significant correlation between the activity in the plasma of diabetics and HbA₁c.
Granulocytes obtained from the patients were destroyed by supersonic waves, followed by centrifugation. The supernatant was used an enzyme solution. Protein content in the enzyme solution was measured by the method of Lowry. The level of enzyme activity in the granulocytes was expressed as absorbance value/h/pg protein. The levels of enzyme activity in the granulocytes of diabetics (4.68×10^-3±1.85±10^-3/h/μg protein) were significantly (p<0.05) less than the enzyme activity in the granulocytes of non-diabetics (5.56×10^-3±1.76×10^-3/h/μg protein). There was no significant difference between or among the levels of enzyme activity in the granulocytes of diabetic groups classified according to the types of therapy given the presence of proteinuria and the presence or grade of retinopathy.
Previosuly we observed that enzyme activity in the kidney cortices of streptozotocin-treated diabetic rats was significantly less than that in the kidney cortices of control rats. The enzyme activity in the plasma of diabetic rats was significantly greater than that in the plasma of control rats.
It may be that the activity of elastase as a collagen-hydrolytic enzyme in the glomerulus of diabetics is low. Such a low content of elastase activity in the glomerulus leads to thickness of the basement membrane and subsequent diabetic glomerulosclerosis.

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Serum levels of apolipoprotein (apo C-II and apo E) and lipids (total cholesterol: TC, triglyceride: TG) were studied in 54 patients with non-insulin-dependent diabetes and 54 normal controls. The diabetics consisted of 29 patients on dietary treatment alone (diet group), 12 patients treated with oral hypoglycemic agents (oral agent group) and 13 patients treated with insulin (insulin group). The levels of apo C-IT and apo E were measured by the single radial immunodiffusion (SRID) method.
The following results were obtained:
1) The serum levels of apo C-II and apo E were correlated significantly with the serum levels of TC and TG, respectively.
2) Positive correlation was observed between apo C-II and apo E level.
3) The serum levels of apo C-II and apo E in the diabetics were significantly higher than those in the controls.
4) The diabetics were further divided into four subgroups based on serum TC and TG levels. The serum levels of apo C-II and apo E in patients with normolipidemia were not significantly elevated as compared with those in the controls The serum levels of apo C-II and apo E in patients with hypercholesterolemia (TC>220mg/dl) and/or hypertriglyceridemia (TG>150mg/dl) were significantly elevated as compared with those in patients with normolipidemia. Especially in patieents with hypertriglyceridemia, the serum levels of apo C-II and apo E were markedly elevated.
These results suggest that in the defect of hypertriglyceridemia, VLDL-TG and VLDL remnant catabolism may contribute to the elevation of serum apo C-II and apo E levels in diabetics.

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Eleven newly-onset type I diabetics (aged from 13 to 34 yers, 5 males and 6 females) were treated with an artificial endocrine pancreas (Biostator®) for 24 to 48 hours. Based on the data obtained, an intensive insulin therapy composed of a combination of short-acting and intermediateacting insulins with selfmeasurement of blood glucose was introduced to each patient in order to attempt meticulous glycemic control.
Remissions (insulin dose less than 10 units a day) were obtained in 4 cases, of which 2 cases are still in remission after 53 and 11 months respectively. Partial remissions (insulin dose ranging from 11 to 20 units a day) were obtained in 3 cases, of which one case is still in the same condition after 47 months. Four cases did not achieve remission.
The intervals from the onset of diabetes to the application of the artificial endocrine pancreas were within 2 weeks in 3 out of 4 cases with remission, one month in 2 out of 3 cases with partial remission, and more than 2 months in all 4 cases without remission.
Thus, strict glycemic control with an artificial endocrine pancreas and subsequert intensive insulin therapy in the early stages after onset were shown to be effective for obtaining frequent and sustained remissions in newly-onset type I diabetes.

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Urinary N-acetyl-beta-D-glucosaminidase (NAG) activity increases in patients with tubulointerstitial damage. Hb-A₁c and urinary NAG activity were measured every three months for nine months in 24 insulin-dependent diabetics (IDDM) without proteinuria, who were receiving outpatient treatmnt.
1) The values (mean+SE) for NAG activity (U/l) in IDDM patients and control subjects were 19.96+5.38 (at the first point) and 2.15±1.41; for NAG index (U/gCr), 12.68+11.27 and 3.67+0.97, respectively. The values for NAG activity and NAG index were significantly high in IDDM patients (p<0.001). There was significant correlation between fasting blood glucose level and Hb-A₁c (r=0.44, p<0.05). Urinary NAG index was positively correlated with Hb-A₁c in IDDM patients (r=0.66, p<0.001)
2) Although Hb-A₁c and urinary NAG index were measured every three months in IDDM patients, urinary NAG index did not tend to follow the pattern of Hb-A₁c. However, in four cases whose Hb-A₁c levels were under 7% for nine months urinary NAG index varied almost within the normal range in each case. This suggests that urinary NAG index measurement in IDDM patients is important for the management of metabolic control.

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The properties of insulin antibodies in the serum of patients with autoimmune hypoglycemia (6 cases) and insulin-treated diabetes mellitus (13 cases) were comparatively studied.
In the cases of autoimmune hypoglycemia, isotypes of immunoglobulins determined by specific precipitation were IgG and kappa (2 cases), IgG-and kappa-dominant (3 cases) and IgG-and lambda-dominant (1 case).
All of the cases of insulin antibodies had two kinds of affinity sites for porcine insulin as revealed by Scatchard plots and dissociation tests.
Affinity constants of high-affinity sites in the globulin fractions of IgG-and kappa-dominant autoantibodies showed similar values to four kinds of insulin preparations of different origin, porcine, bovine, semisynthetic and native human insulin, but the values were smaller than those of insulin antibodies produced by insulin injection.
Binding affinities of IgG (kappa) and IgG (lambda) fractions isolated from IgG-and kappa-dominant autoantibodies (2 cases) indicated the same binding affinities as those of globulin fractions. Antibodies produced by insulin injection had different binding affinities among three fractions. Furthermore, in the case of IgG-and lambda-dominant autoantibodies, most of the IgG (lambda) and IgG (kappa) fractions were derived from high-and low-affinity sites, respectively.
With regard to dissociation rate constants, there were no significant differences between antibodies obtained from autoimmune hypoglycemia and insulin-treated diabetes mellitus and also among globulin, IgG (kappa) and IgG (lambda) fractions.
In conclusion, insulin autoantibodies were shown to be heterogeneous in their immunoglobulin isotypes and insulin-binding affinities.

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It has been postulated that long-term physical training can improve glucose tolerance and insulin sensitivity. The aim of this study was to estimate the effects of training quantitatively. The tissue sensitivity to exogenous insulin was evaluated in 5 obese diabetics (obesity index: 136.8±5.8%), 7 patients with simple obesity (144.4±7.0%), 10 trained athletes (95.4±1.4%), and 18 controls (94.9±1.8%).
Insulin sensitivity was determined by the euglycemic insulin clamp technique for 120 minutes (insulin infusion plus a variable glucose infusion). The amount of glucose infused (glucose metabolism) is a measure of the overall tissue sensitivity to insulin. In order to adjust the differences of fasting blood glucose levels, glucose metabolic clearance rate (MCR) was calculated.
During the insulin clamp study, comparable plasma insulin levels (70-119μU/ml) were achieved in all groups. Glucose metabolism in the obese diabetics (3.67±0.33mg/kg/min)(p<0.001), and in the patients with smple obesity (3.01±0.21mg/kg/min)(p<0.001), were significantly lower than in the controls (7.46±0.22mg/kg/min), while glucose metabolism in the athletes (11. 63±0.65mg/kg/min) was significantly (p<0.001) higher than in the controls. A strong inverse correlation exists between glucose metabolism and Broca's index (r=-0.883, p<0.001). In the case of MCR, exactly the same tendency was observed. Eight of the obese diabetics continued dietary restriction (1, 000-1, 600 kcal) and physical training for 4-8 weeks. After physical training significant (p<0.005) weight reduction (6.1+1.2kg) was obtained. Glucose metabolism increased from 3.17±0.20mg/kg/min to 4.00±0.35mg/kg/min (p<0.05). MCR also increased from 2.70±0.31ml/kg/min to 4.20±0.55ml/kg/min (p<0.01).
These results suggest that (1) tissue sensitivity to physiological hyperinsulinemia is significantly lower in obese diabetics and (2) proper dietary restriction and physical training can improve decreased insulin sensitivity in obese diabetics.

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The enzyme superoxide dismutase (SOD; superoxide oxidoreductase, EC1. 15. 1.1) is a scavenger of the superoxide radical, a toxic species that has been implicated in DNA damage and protein sulfhydryl oxidation. There are many reports currently available concerning the pathological significance of the superoxide radical on Type 1 diabetes. Therefore, we have studied SOD activities in erythrocytes from non-obese diabetic (NOD) mice, which display a syndrome showing dramatic clinical and pathological features similar to those of Type 1 diabetes in man.
SOD activity in mouse erythrocytes was measured by the nitrite method. This method was 10 times more sensitive than the conventional cytochromec method, and the accuracy and the recovery of SOD activities obtained from this method showed respectable results. Erythrocyte SOD activities in NOD mice were always high in comparison with those in control ICR mice from 3 to 13 weeks of age. SOD activites in NOD mice increased at 6 weeks of age, rapidly decreased around 8 weeks of age, rose again up to 11 weeks of age and then decreased thereafter. Islet cell surface antibodies (ICSA) determined by protin A radioligand assay, were demonstrated at 7 and 11 weeks of age in NOD mice.
These results suggest that the changes in SOD activities between 6 and 9 weeks of age may contribute to the appearance of circulating ICSA. Since changes in the activity of this protective enzyme reflect the amount of the superoxide radical, the latter may play an important role in the pathogenesis of Type 1 diabetes.

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Using an indirect immunofluorescence test on living cultures of JHPI-1, an easier method for detecting islet cell surface antibodies (ICSA) was developed, and the reproducibility of this method was examined.
Human pancreatic B-cell clones (JHPI-1) employed as target cells of ICSA were cultured at a density of 104 cells per well in 96-well microculture plates before use. Heat-inactivated, 1:4-diluted sera were incubated with the JHPI-1 cells for 1 hour at 37°C. After the cultures had been washed twice, FITC antihuman IgG serum was added to each well, and incubated for 1 hour at 37°C. ICSA were examined using an inverted microscope equipped with an epifluorescence condenser. The prevalence of ICSA in this method for IDDM was signficantly (p<O.01) correlated with that of Lernmark's original method. The inter-observer and inter-assay variation showed good reproducibility (p<0.01).
According to this method, the prevalence of ICSA was 38.6%(39/101) in sera from IDDM patiens. In conclusion, an easier method was developed for detecting ICSA using monolyers of JHPI-1 as target cells. This new method, with a good reproducibility, is useful for detecting ICSA.

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The absorption of monocomponent semisynthetic human insulin (SHI) was compared with that of purified porcine insulin (PPI) by determining serum insulin levels after subcutaneous injection of Actrapid, NPH and Monotard preparations. The studies were undertaken in 36 newly insulin-treated diabetic patients.
1. The levels of serum should be willed out IRI sut in after injection of Actrapid SHI were similar to those after PPI injection when 8 units of insulin were administered. Maximal IRI was observed 2 hours after injection. Blood glucose levels at 3 hours after injection were significantly lower after SHI than after PPI administration (p<0.05). When 12 units of insulin were given the absorption of SHI was more rapid than that of PPI, assessed by measurement of serum IRI, increment from basal IRI (JIRI) and total increment (IJIRI) four hours after injection (p<0.05). Correspondingly, the blood glucose curve was significantly lower after SHI than after PPI injection (p<0.05).
2. Daily profiles of blood glucose and IRI showed no differences between the two NPH preparations.
3. The serum IRI after injection of Monotard SHI rose more rapidly than PPI. The maximal IRI was reached after lunch and before the evening meal, following injection of Monotard PPI.
These results indicate that the absorption of intermediate-acting SHI is indistinguishable from that of PPI, but short-acting SHI is absorbed more rapidly from subcutaneous tissue than PPI. It is therefore suggested that the bioavailability of human insulin differs from that of porcine insulin.

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Non-obese diabetic (NOD) mice aged 30 to 60 days were treated orally with Cyclosporin (Cs) at doses of 25, 15 and 2.5mg/kg every other day until 160 days of age. Diabetes developed in 12 out of 18 non-treated control mice (67%) with partial to complete Langerhans' islet destruction associated with remarkable lymphocytic infiltration and the pancreatic insulin content of diabetic nontreated NOD mice was remarkably decreased compared with that of Cs-treated mice. In contrast, Cs-treated NOD mice showed neither clear increment of plasma glucose levels nor development of insulitis.
Moreover on day 160, 6 out of the Cs-treated mice were taken off Cs, but by day 280, none of these mice in which Cs had been discontinued had become diabetic.
Subsequent Cs treatment was started after development of glucose intolerance. Twenty-five mg/kg of Cs was administered every other day for 35 days. Eight out of ten mice (80%) treated with Cs developed severe hyperglycemia and progression of glucose intolerance.
Cs therefore appeared to have preventive effects but few therapeutic effects on diabetes in NOD mice.

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Transient hyperopic changes were observed in two adult-type uncontrolled diabetics. Case 1: A 53-year-old man with diabetes mellitus of 5 years' duration was admitted to our hospital for regulation of diabetes. He was treated with diet and exercise therapy. On the 7th hospital day, he noticed he could not see well with his glasses (right-2.75 D, left-3.0 D). On the 17th hospital day, his refraction revealed hyperopic change: V. d., 1.0 with+0.75 D-cyl-0.75 D Ax. 40°; V. s., 1.0 with+0.75 D-cyl-0.5 D Ax. 140°. Case 2: A 56-year-old woman with diabetes of 3 years' duration was admitted to our hospital, and was treated with glibenclamide (2.5mg/1×d) and dietary therapy. On the 4th hospital day, she noticed she could not see well with her glasses (right-1.5 D-cyl-0.75 D Ax. 135° left-1.25 D-cyl-0.75 D Ax. 50°). On the 18th hospital day, her refraction revealed hyperopic change: V. d., 0.9 with+0.5 D-cyl-1.0 D Ax. 135°; V. s., 0.9 with+0.5 D-cyl-1.0D Ax. 50°. These hyperopic changes were improved after blood glucose levels had become normalized. Eye glasses should not be prescribed for diabetics unless their blood glucose is controlled and stabilized.

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