Partially purified porcine proinsulin C-peptide (porcine C-peptide) was rapidly injected intraveously into 7 pigs together with porcine insulin and plasma concentrations of both peptides were assayed separately by highly specific radioimmunoassay techniques. The average immunological half-life of porcine C-peptide and porcine insulin were calculated as 9.9±0.5 min and 8.2 ± 0.3 min (M±SD), respectively. Thus, porcine C-peptide was found to have a half-life longer than that of insulin. On a molar basis this may account for a plasma concent ration of porcine C-peptide higher than that of insulin in the pig.
Recoveries of added porcine C-peptide, proinsulin and insulin from porcine serum charcoal treated with acid alchohol extraction were calculated as 33.4±3.9, 67.4 ±5.4 and 74.9±3.8%(M SD), respectively.
Serial plasma samples from C-peptide injected pigs were extracted by acid ethanol and assayed for C-peptide and insulin immunoreactivities after gel filtrations (Bio-Gel P-30, 1.2 ×90 cm, 3 M Acetic Acid). The major peaks of C-peptide and insulin immunoreactivities were eluted in the Cpeptide and insulin areas of Bio-Gel columns. The other immunological activities of C-peptide or insulin were not revealed in the fractions corresponding to molecules smaller than these peptides.

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The incidence of abnormal glucose tolerance (diabetic and borderline pattern) on multiple health checking screened by diagnostic criteria for diabetes suggested by Japan Diabetic Society of 1970 was generally high: half or more of the subjects. A study was made to determine statistically more practical screening level for oral 50 g glucose tolerance test (50 g GTT).
The 50 g GTT was carried out on 1191 Hiroshima University staff members, 987 males and 204 females aged from 24 to 63 years, during 1971 through 1974. Venous serum glucose at three time points, fasting, and 1 hour and 2 hours after 50 g glucose loading was determined with the o-toluidine boric acid method.
On the basis of a distribution curve of the glucose level, the 97.5 and 84.0 percentile values were obtained by a graphical method with normal and logarhythmic probabilty paper. These values were considered to be abnormal (diabetic) and normal (non-diabetic) points respectively, and the remainder probably abnormal (borderline).
The critical serum glucose levels for diabetic screening recommended by this study were 120 mg/ dl at fasting, 220 mg/dl at 1 hour and 140 mg/dl at 2 hours on 50 g GTT, and normal values were below 110 mg/dl, 180 mg/dl and 120 mg/dl, respectively.
The validity of this new criteria for diabetic screening can be determined by further observation and follow up study.

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To clarify the interrelationship between endocrine and exocrine pancreatic functions, external pancreatic secretions and plasma pancreatic enzyme responses to pancreozymin-secretin were analyzed in 57 primary diabetic subjects. The effects of insulin on exocrine pancreatic functions were also studied in streptozotocin-diabetic rats.
1. Volume, maximum bicarbonate concentration and enzyme output (amylase, trypsin and lipase) of duodenal contents obtained after pancreozymin-secretin (P-S) stimulation decreased significantly in diabetic subjects when compared to normal subjects. Among these indices, the most marked abnormalities were observed in enzyme output, especially in amylase output. Low amylase output was found more, frequently in poorly controlled diabetic subjects.
2. Positive plasma enzyme responses to P-S were observed more frequently in poorly controlled subjects, judged as positive when one or more of the three enzymes were provoked. Positive plasma enzyme responses found in poorly controlled diabetic states were markedly improved after insulin treatment. Abnormally high plasma amylase and lipase levels observed in diabetic ketoacidotic coma were also reduced to normal levels after the metabolic derangement was improved by insulin.
3. In streptozotocin-diabetic rats, both acute and chronic, only amylase output in duodenal contents to P-S stimulation decreased significantly when compared to normal rats, as a result of the very low amylase content in the pancreatic tissues. The plasma amylase level was low an
was not changed significantly by P-S stimulation. Plasma lipase, on the other hand, was easily provoked in diabetic rats. Such abnormalities as low amylase content, low amylase output, low plasma amylase level, and positive plasma lipase response were restored to normal after insulin treatment.
These findings indicate that insulin plays an important role in maintaining normal exocrine pancreatic function. These exocrine pancreatic dysfunctions secondary to diabetes mellitus are discussedas “diabetic exocrine-pancreatopathy”.

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With the use of a commercially available radioimmunoassay kit for blood C-peptide, the assay of urinary C-peptide was conducted. Urine C-peptide immunoreactivity (CPR) could be assayed adequately after dilution of urine to 1: 20-1: 40, as revealed by dilution and recovery tests. Urine CPR was stable for 1-2 days at room temperature, and as long as one year if stored frozen at-20°C. Urine CPR was eluted by gel filtration as a single peak in the region where 125I-labeled C-peptide was eluted. In a patient with an insulin antibody, gel filtration of serum revealed a peak of CPR in the region of higher molecular weight, but urine CPR was eluted in the normal region.
Normal subjects excreted 1.2±0.6 pg CPR per hour in urine at fasting. After oral administration of 50 g glucose, urine CPR increased three-fold, approximately paralleling the change in serum CPR. In diabetic patients, the increase in urine CPR after glucose load was sluggish and small, and in juvenile-type diabetic patients urine CPR response was virtually absent. Normal CPR clearance was 7.1±2.5 ml/min. 24 hour urinary excretion of CPR in norma subjects was 92.2±38.6 μg/kg, 1.52±0.55 μg/kg body weight, and 55.1±18.2 μg/mg creatinine. It was extremely low in juvenile-type diabetics, slightly or moderately decreased in non-obese adult-type diabetics, but variable in obese diabetic patients. Urine CPR was not detected in a patient who underwent total pancreatectomy. Patients on steroid treatment for various blood and other disorders had high fasting serum CPR levels, and excreted excessive amounts of CPR in the urine. In these patients, there were highly significant correlations between 24 hour urine CPR and fasting serum CPR or serum CPR response during the glucose tolerance test. In renal insufficiency, the fasting serum CPR was high, but urinary CPR excretion was decreased. Three insulinoma patients had elevated fasting serum CPR, but their 24 hour urines contained normal amounts of CPR. Despite some limitations in these special cases, urinary CPR can be used as an additional indicator of endocrine pancreatic function.

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It has been generally accepted that loss of body weight is difficult in obese diabetic patients treated with sulfonylurea drugs. But there are few articles about investigation of the change of body weight during long-term treatment with sulfonylurea drugs in these patients.
In this paper we report our study of the changes of body weight, fasting blood sugar and total cholesterol in 36 obese patients treated with sulfonylurea drugs (Su-group) for from three to six years and compared the results with the results obtained in 24 obese subjects treated with diet only (D-group).
Mean body weight in the Su-group was reduced in a way similar to that in the D-group for six years. Especially in the first three months loss of body weight was remarcable in both groups, but was more effective in the D-group. The levels of fasting blood sugar and total cholesterol in the Su-group were kept within normal range during these six year's treatment.

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The prevalence of diabetic retinopathy and its correlation with the serum HGH levels were studied in the aged with diabetic glucose tolerance (diabetics). The following results were obtained:
1. Diabetic retinopathy was found in 37 % of the aged diabetics. Most of the retinopathies (96%) were non-proliferative and were not severe (ScottII>, 67 %).
2.0ccurrence of diabetic retinopathy correlated well with the duration of abnormal glucose tolerance, but not with the age of the patient.
3. Basal serum HGH levels in the aged diabetics with retinopathy was not significantly different from those without retinopathy. The maximum response of HGH to insulin hypoglycemia was significantly lower in diabetics with retinopathy than in those without retinopathy. On the other hand, HGH responses to arginine in these two groups were not significantly different.
Although a high serum HGH level has been postulated as a causative factor in the development of diabetic retinopathy, particularly the proliferative type, in the young, our present study clearly demonstrates that HGH may not play an important role in the development of retinopathy in aged diabetics. Low levels of serum HGH in aged diabetics may be related to a non-proliferative type of retinopathy.

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Diabetic neuropathy was studied in alloxanized diabetic rats, using the method of motor nerve conduction velocity (MCV) and teased fiber stuby.
The MCV was measured in the tail nerve with needle electrodes. Preliminary experiments showed that the optimal age of rats was greater than 180 days. A stable temperature of 37°C was obtained by dipping a tail in a liquid paraffin bath for more than 5 minutes.
In the teased fiber study, biopsy specimens of the peroneal, sural nerve and the root of sciatic nerve were used. These biopsy specimens were fixed with 2 % glutaraldehyde solution at pH 7.4. Teasing of nerves was performed after osmication with 1 % osmium tetroxide.
Alloxanized diabetic rats were divided into five groups according to the course before and after the administration of alloxan, that is, before administration and 1 week, 2 weeks, 3 weeks and 4 weeks after administration. The results from these groups were compared with those from untreated control groups.
The MCV in the diabetic rats of the groups of 2, 3 and 4 weeks was significantly lower than the MCV in untreated control rats.
In the teased fiber study, a significant difference was observed between diabetic rats in the groupsof 3 and 4 weeks and untreated control rats. The main change was segmental demyelination in the distal portion of the sural nerve.
In the group of 4 weeks, the finding of the remyelination in the damaged nerves was observed.

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In our previous report, we demonstrated that complements such as β₁-C/A-globulin (C3) and β₁- E-globulin (C4) increased significantly in the serum of patients with diabetes mellitus.
In this study, we analyzed the hemolytic activity of the serum complement. We also measured various components of the whole complement system and the complement-inhibitor in the serum of the diabetics.
CHSO was measured by Mayer's original method in 95 adult-onset diabetics and Ciq, C1s, C3a, C3A, C5, and C9, as well as C1s-INH were measured quantitatively by the single radial immunodiffusion method using Behringwerke's Partigens in 32 adult-onset diabetics. The serum of 20 healthy adults served as the control. All of the diabetics (group DM) were divided into three groups; group D diabetics without clinical manifestations of microangiopathy, group R: diabetics with retinopathy, and group N: diabetics with nephropathy.
The following results were summarized. (1) CH_5O increased in all diabetic groups. Its increase was more predominant in group R than in group D. In group N, CHSO showed changes: both increase and decrease. (2) Among the early-acting components, increase of C1q was minimum but Cis showed significant increase in group DM, and R. (3) C_1s-INH showed a significant increase in all diabetic groups. Increase of C_1s-INH became more pronounced as involvement of microangiopathy progressed. (4) Among the complements related with an alternative pathway, C₃a did not show any remarkable change and C₃A showed minimal increase in the diabetic groups. (5) Among the late-acting components, C₅ showed remarkable increases in all diabetic groups and C₉ showed moderate increase along with the progression of microangiopathy.
From our previous and present studies, it is thus clearly confirmed that hemolytic activity of the complement, almost all components of the complement system as well as complementinhibitor were generally increased in diabetics, particularly in those with microangiopathy. Although the pathophysiological nature of the increase of the serum complement system in diabetics is still obscure, it is suggested that there are possibilities that these changes of the complement system might be reflecting the presence of insidious inflammatory process of the slightest degree in the diabetics, and also that changes of the complement system might be playing a very important role in the development of microangiopathy as the complement system is recently found to be closely related to the coagulation and fibrinolysis systems as well as the kinin system in the blood.

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The influence of obesity (% relative body weight, RBW) and fasting serum triglyceride level (TG) upon other metabolic derangements such as fasting serum immunoreactive insulin level (IRI), fasting blood sugar level (FBS), and insulinogenic index (II) were studied in 606 Japanese adults with multiple regression analysis (stepwise forward selecting method) by computer system.
93 subjects of normo-TG (TG < 100 mg/dl) with normal obesity (RBW < 100 %) had little possibility of increasing in IRI or FBS, but 24 subjects with normo-TG and high obesity (RBW > 120 %) had some possibility of becoming hype-IRI with normo-FBS.
Hyper-TG (TG > 250 mg/dl) subjects with or without obesity (n=22, 12) had, however, a high possibility becoming hyper-FBS and hyper-IRI, independently of obestiy.
Obesity is known as a parameter of insulin insensitivity. The present findings suggest that the increase in serum TG level also possibly indicates another parameter of insulin insensitivity, because hyper-TG with normal body weight revealed increases in serum IRI and FBS levels.

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A case of diabetes with severe insulin resistance and Sjögren's syndrome was reported by M. Kihata and co-workers in 1973. In 1975, Flier, Kahn and co-workers reported a method for detection of anti-insulin receptor antibodies, and demonstrated the presence of anti-insulin receptor antibodies in the serum of this patient, using circulating human monocytes and cultured human lymphocytes. We attempted to prove the presence of anti-insulin receptor antibodies in the serum of the same patient, using pellets of insulin receptors obtained from human placental tissues (human placental membrane), and obtained the following results.
1) Although this patient had been injected with several thousand units insulin without hypoglycemia, the serum was negative for anti-insulin antibodies and the serum immunoreactiveinsulin (IRI) was 960μu/ml.
2) The binding of ¹²⁵I-insulin to human placental membrane preincubated by the patient serum was inhibited.
3) Also the binding of ¹²⁵I-insulin to human placental membrane was markedly inhibited by the protein fraction of the serum eluted with the Sephadex G-50 column.
4) The immunoglobulin class of this protein fraction which inhibited binding was IgG.
It is suggested that the use of human placental membrane for the detection of anti-insulin receptor antibodies in the serum would be possible.

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In order to investigate the glucoreceptor mechanism involved in insulin release and the haracter, characterics of the mechanism we have studied the dynamics of insulin release from the perifusd isolated islets of rats under slow-rise stimulation with α and β-D-glucose (α and β anomers), which were kept at 4°C in solution to prevent anomerization we tried to analyze kinetically each dose response curve obtained from those dynamics.
At concentrations ranging from 5 to 9 mM greater release of insulin was stimulated by the β anomer than by the β anomer. Both dose response curves were sigmoid with the same maximal rate of insulin release (0.9μu/ml/islet/min) at around 11 mM glucose, but the value of Km for the, β anomer was higher than that (6-7 mM) for the α anomer with 2 mM glucose.
Since the Lineweaver-Burk, plots for the α and β anomers were not straight lines, but parabolic ones, those insulin responses seem to be represented as n-ordered reactions (n> 1).
The logalithmic values were -3.9±0.7 and -5.9±0.6 (mM^-n, M±S. E. M.), respectively for the α and β anomers, and therefoer, the affinity of the glucorecepter for the α anomer seemed to be one hundred times as strong as that for the β anomer.
The Hill plot for the α anomer was represented as a single line (n=4.8), whereas that for the β anomer consisted of two straight lines (n=2.3 and 6.2) which had a breakdown at 8.3 mM glucose. Therefore, this result shows that the recepter-recepter interaction may be increased at concentrations of the β anomer over 8.3 mM.
In conclusion, we have been led to the following suggestions: that the pancreatic B cells contain common glucoreceptors on the plasma membrane permitted to bind both anomers of D-glucose, and that the substrate-recepter complex formed between the β anomer and glucoreceptor may be “an incompleted binding” to induce an insulin release less than that with the α anomer at concentrations lower than 8.3 mM. But, in accordance with the raising of the β anomer concentration, the β anomer caused the same amount of insulin release as the α anomer through a change of the glucoreceptor conformation.

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Two cases of diabetes poorly controlled developing chylomicronemia were presented.
The first case, a 16-year-old female, was admitted because of ketoacidotic semicoma. On admission, her serum had a milky appearance, and contained a very high concentration of triglyceride (over 4, 350 mg per 100ml). The concentration of serum cholesterol was 197 mg per 100ml. The agar gel electrophoretic pattern of lipoproteins demonstrated 4% of chylomicron, 19%β lipoprotein, 62% pre-β lipoprotein, and 16%α lipoprotein. The milky serum became clear as the patient recovered from coma on the day after injection of insulin.
The second case was a 43-year-old male acromegalic patient with diabetes. Since the pituitary surgery 2 years ago, the patient has been treated with medroxyprogesterone and a sulfonylurea drug in addition to a maintenance dose of hydrocortisone. During this regimen the diabetic state deteriorated rapidly due to his neglect of caloric restriction, and laboratory evidence suggestive of liver injury was noted. The blood sugar level was 424 mg per 100 ml, growth hormone 53 ng per ml, triglyceride 1, 950mg per 100 ml, and cholesterol 298 mg per 100 ml. Agar gel electrophoresis of serum lipoproteins showed 19% chylomicron, 28%β lipoprotein, 45% pre-β lipoprotein, and 8%α lipoprotein. Withdrawal of all drugs and strict dietary restriction resulted in prompt improvement of the hypertriglyceridemia and in good control of the diabetes.
As regards the biochemical sequence of chylomicronemia occurring in these cases, it is assumed that a primary event for the deterioration of diabetic states and lipid metabolism is insulin insufficiency which brings on a disturbance in the removal of serum lipids, and in turn hypertriglyceridemia, chiefly by reduction of lipoprotein lipase activity.

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