Eight healthy subjects were studied 3 times serially at 1 week intervals. Blood glucose and plasma insulin were investigated for 240 minutes after oral administration of 40, 80 or 120 mg of gliclazide. Blood glucose fell significantly with all of these doses of gliclazide. The decrease in blood glucose induced by the administration of gliclazide was significnatly greater than that observed in the control group without the drug. Plasma insulin rose significantly after the administration of either 80 or 120 mg of the drug, while the mean increment of plasma insulin was not remarkable in the case of 40 mg, probably because of the dilution by systemic circulation. It was demonstrated that both the maximum increase in insulin and the maximum decrease in blood glucose were pro. portional to the dosage of gliclazide.
Experimental studies were performed using a preparation of in situ local circulation of the pancreas in dogs. Gliclazide was infused into the superior pancreatic artery in amount of 10, 20 or 40 mg for 10 minutes. The plasma levels of insulin and glucagon were assayed in the blood obtained from the pancreatic vein and the blood glucose levels in the femoral artery were determined simultaneously, A significant rise of plasma insulin was observed with either dose of gliclazide, but no remerkable change was observed in the glucagon or in the blood glucose. Thus, the following were confirmed: 1) gliclazide has hypoglycemic action in man, 2) gliclazide causes insulin release in man and dogs, and 3) gliclazide does not inhibit the secretion of pancreatic glucagon in dogs. Gliclazide is one oi the unquestionable hypoglycemic agents and this action is thought to be brought about mainly 133, insulin release in response to the drug, although our results cannot exclude any extrapancreatic action of the crug.

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A radioreceptor assay (RRA), similar to the bioassay that is more essential than radioimmunoassay (RIA), has recently been studied as a measurement of insulin in serum. A sensitive method has been developed for measuring insulin in serum using an insulin receptor solubilized from normnl human placental membranes.
This receptor assay system is specific for insulin and has a sensitivity similar to that of the current radioimmunoassay system.
There was a high degree of correlation between the results of immunoassay and receptor assay over the physiological range of the normal serum insulin level.

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Diabetic neuropathy was studied histologically in alloxanized diabetic rats, with the method of motor nerve conduction velocity (MCV) and with the quantitation of myelinated nerve fibers.
The MCV was measured in the tail nerve with needle electrodes. Biopsy specimens were collected from the proximal portion (peroneal-proximal) and distal portion (peroneal-distal) of the peroneal nerve, the proximal portion (sural-proximal) and distal portion (sural-distal) of the sural nerve, and the motor root (motor-root) and sensory root (sensory-root) of the sciatic nerve.
On the transverse section of these nerves, the density of the total number of myelinated nerve fibers (number of nerve fibers/mm²) was calculated, and histograms of myelinated nerve fibers according to a diameter were made.
Diabetic rats were divided into five groups according to the course: before, and 1, 2, 3 and 4 weeks after administration of alloxan. The results from these groups were compared with those from untreated control groups. The MCV in the diabetic rats of the 2, 3 and 4 week groups was significantly lower than that in the untreated control groups. A significant decrease of the density of total myelinated nerve fibers was observed in the sural-proximal and sural-distal in the 3-week groups as well as in the peroneal-proximal, peroneal-distal, sural-proximal, sural-distal and sensory-root in the 4 week group in the diabetic rats. A significant decrease in the number of myelinated nerve fibers and disappearance of the peak in the myelinated nerve fibers of large diameter were observed in the peroneal and sural nerve, especially in the sural-distal in the 2, 3 and 4 week groups in the diabetic rats. There was no change in the motor-root, on the other hand, in the sensory-root, decrease of myelinated nerve fibers of large diameter was characteristic, although the number of myelinated nerve fibers of the small diameter remained unchanged in the diabetic rats.
It is considered that these findings are valid for investigating the pathogenesis of human diabetic neuropathy.

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C-peptide responses to glucose were investigated in full term fetuses at the time of delivery. Glucose was administered continuously by drip infusion to the mothers during labour. Plasma glucose of the umbilical artery and vein rose immediately during a glucose load, whereas plasma IRI and C-peptide of these vessels were not elevated during first 40 minutes of the glucose load. Significant elevation of plasma IRI and C-peptide was demonstrated only after 44 minutes or longer. It was also demonstrated that placental transfer of C-peptide is extremely limited, as no significant correlation between the levels of umbilical arterio-venous difference and maternal vein levels was found. Blood samples of these fetuses were also obtained by heel stab 30 minutes after birth. IRI levels were significantly lower during this period, while C-peptide levels were elevated.
IRI and C-peptide responses in newbornes and older infants were examined by administering milk (15 ml per kg body weight). Their responses varied quite widely and seemed not to be dependent on the age of newborn. Relatively high C-peptide levels were found in 1-4 day-old infants. These findings may be partly due to immaturity of renal function during this postnatal period.

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Monocomponent insulin (MC insulin) was evaluated by comparing its antigenicity with that of conventional insulin in diabetics under various conditions, such as the presence of insulin allergy, newly diagnosed insulin dependent cases, or diabetics undr long term treatmert with conventional insulin.
In the course of MC insulin treatment of patients, the antigenicity was estimated by the change of insulin binding antibody and the insulin requirement.
No immunogenic effect was observed in the newly diagnosed diabetics during 6-12 month MC insulin treatment.
In 66.7 per cent of the long-term treated diabetics who had switched to MC insulin from conventional insulin, antibody levels showed a tendency to decrease (5-31%; mean 16.6%) but did not reach normal levels.
In other diabetics, antibody levels either did not change or showed a slight increase (4 %).
All insulin allergy disappeared after switching to MC insulin treatment.
In 88.9 per cent of the long-term treated diabetics after switching to MC insulin, the insulin requirement showed a tendency to decrease (4-28 U/day ; mean 16.5 U/day).
We suggest that MC insulin may be less antigenic than conventional insulin and more effective for the treatment of insulin resistant diabetics and diabetics allergic to insulin.

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The sera of 69 parents and 9 siblings of 57 patients with diabetes mellitus of childhood and the controls matched for age and sex, were tested by the thyroglobulin and microsome-coated tanned red cell hemagglutination test (Fuji Zoki Co. Tokyo).
1) Diabetes mellitus in childhood (probands):
One of 79 children with diabetes mellitus (1.2%) had antithyroglobulin antibodies and fifteen (19%) had antimicrosomal antibodies compared with 0.4% and 1.1% respectively in 437 disease controls.
2) Parents of probands:
The incidence of antithyroglobulin and antimicrosomal antibodies was 5.8% and 20.2% respectively.
Four of 53 mothers (7.5%) had antithyroglobulin antibodies and thirteen (24.5%) had antimicrosomal antibodies compared with 3.1% and 4.1% respectively in 538 women controls (30-49 years).
One of 16 fathers showed a positive result with antimicrosomal antibodies.
3) iblings of probands:
One of the siblings had antimicrosomal antibodies.
4) Parents and siblings of probands having positive thyroid antibodies:
Five of them (41.6%) had antimicrosomal antibodies.
5) Parents and siblings of probands with negative thyroid antibodies:
The incidence of antithyroglobulin and antimicrosomal antibodies was 4.5% and 15.1% respective y.
These findings suggest that immunogenetic factors may be responsible for some cases of diabetes mellitus in childhood.

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In order to clarify the characteristics of the cell membrane in insulin release, the effect of trypsin treatment on the release of insulin from islets of Langerhans isolated from rat pancreas was studied.
Pancreatic islets were isolated from male Wistar rats by the conventional collagenase method. The islets were pretreated with 1 mg/ml trypsin for 60 min in a vial containing 0.5 ml of Krebs Henseleit bicarbonate buffer (KHBB), 5 mg/ml bovine serum albumin and 3.3 mM glucose under he gas phase of 95% 02-5% CO₂.
After rinsing 3 times in 5 ml of KHBB, 5 islets were transferred to an incubation vessel containing various agents and were incubated for 60 min. Insulin released in the second incubation medium and insulin contained in the islets were determined by double antibody radioimmunoassay. The islets were frozen at -80° immediately after incubation, and freeze-dried at -40°, 0.001 mmHg. Islet ATP and glucose were assayed by the enzymatic cycling method.
Results were summarized as follows;
1. Pretreatment with various concentrations of trypsin (0.01 to 10mg/ml) for 5, 15, 30, 60 or 90 min brought about a tendency to suppress 16.7 mM glucose-induced release of insulin from the islets. Significant decrease of insulin release was observed as a result of treatment with 1 mg/ml trypsin for 30 min. For assurance, treatment with 1mg/ml of trypsin for 60 min was adopted in successive xperiments.
2. The insulin content of trypsin-pretreated islets was not modified significantly compared with that of non-treated control groups. Furthermore, treatment with trypsin did not change the glucose or ATP concentration of the islet.
3. Trypsin slightly increased the basal level of insulin release, while high concentrations of glucose significantly reduced insulin response.
4. The decrement of 16.7 mM glucose-induced insulin release by trypsin treatment was restored to a certain degree by addition of adenosine, glucagon or db-cAMP, but not to the level of insulin release by these agents from the trypsin non-pretreated islets.
5. The observations mentioned above suggest that:
i) Trypsin modified a process other than the intra-islet insulin synthesis and glucose metabolism; possibly the B-cell membrance which has been thought to be associated with the site of glucoreceptor and/or final site of insulin release. The latter was affected very slightly by trypsin because there was no difference between the insulin contents of trypsin-treated and nontreated islets.
ii) Intracellular events were necessary to maintain function of islet cells, especially insulin release, while incomplete restoration showed the importance of the cell membrane, possiblly thatof the glucoreceptor in insulin release as well.
Further studies are needed to clarify the exact mechanism of trypsin action and thereby the significance of the B-cell memberance as a glucoreceptor in insulin release.

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A 44-year-old man was admitted to the hospital in November 1975 because of an attack of hy poglycemia. The fasting blood glucose, mmunoreactive insulin (IRI) and C-peptide immunoreactivity (CPR) levels were 41-48 mg/dl, 37-71.5 AU/ml and 3.15-5.00 ng/ml, respectively. The blood glucose levels during the hypoglycemic attacks were 19-41 mg/dl. During oral 50 g glucose loading' serum IRI and CPR reached the peak, at 30 min, 496.0 μU/m1 and 12.20 ng/ml, respectively, and intravenous tolbutamide or oral L-leucine administration caused hypoglycemic attack resulting in loss of conciousness and convulsion.
ngiography of the celiac artery revealed the presence of pancreatic tumors, which were resected-Two tumors 1.5×1.25 cm and 1.75×1.0 cm were found at the tail of pancreas. The histological diagnosis was a benign insulinoma.
To investigate the autonomy of insulin secretion from insulinoma, insulin suppression tests were performed administering fish insulin, somatostatin and propranolol-adrenalin, respectively. In the administration of fish insulin, CPR levels, an indicator of endogenous insulin secretion, were not completely suppressed by the induced hypoglycemia. However, somatostatin and propranolol-adrenalin, which have been reported to have a direct suppressive effect on insulin release from the β Cell, suppressed the IRI and CPR.
These results suggest that insulin secretion in this patient was not controlled by blood glucose levels, though it was directly suppressed by somatostatin and propranolol-adrenalin. Therefore, in some cases of insulinoma the suppressive test with exogenously administered insulin may be diagnostically more useful than the stimulatory tests which sometimes have false negative or false positive results.
The serum levels of proinsulin measured by the method or Melani were high before resection of the insulinoma but could not be detected after the operation. The fact indicated that proinsulin was secreted from the insulinoma and that its measurement is important for diagnosing insulinoma.

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Very few cases have been reported which showed only meningeal symptoms during diabetic coma. A 43year old house wife who had no history of diabetes suffered from influenza on 10th of Oct.1974, and gradually began complaining of thirst and nausea and epigastralgia which radiated to the back by Oct. 20 th. She was admitted to the hospital because of somnolence and tremor of the hand. Meningitis or subdural bleeding was suspected from the prevalent signs of nuchal rigidity and Kernig's sign, but was denied by the CSF findings. Muscular defense was noted on her epigastrium. Laboratory examination revealed that the serum amylase was 3420 IU/L, urinary acetone negative, hematocrit 55%, blood sugar 1570 mg/dl, serum Na 149 mEq/L, serum K 4.5 mEq/L, BUN 62.5 mg/dl, serum osmotic pressure 508 mOs/L, pH 7.325 and BE-5.0 mEq/L. From the above data, her condition was diagnosed as non-ketotic hyperosmolar diabetic coma due to acute pancreatitis.
Although the mechanism of such meningeal symptoms during diabetic coma have not been completelyelucidated, dehydration must have been the trigger, because the symptom disappeared 23 hours after infusion of 8000 ml of transfusion fluid.

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In Japan, since 1970, fourteen patients have been reported who suffered from a new type of hypoglycemia, called insulin autoimmune syndrome, associated with insulin antibodies without any immunization using insulin preparations. One of characteristics of this syndrome is that remission of the disease occurs pontaneously.
A 70-year-old Japanese man was admitted to the Saiseikai Imabari Hospital on February 12, 1975 because he became unconscious every morning after February 5. Each attack was treated with intravenous injection of glucose. Attacks occurred in the early morning of February 13 and 14. Blood glucose during the two attacks was 40 mg/100ml and 24 mg/100ml respectively. On February 15 the attacks disappeared spontaneously, and hypoglycemic attacks occurred only once in ten days. Immunoreactive insulin (IRI) extracted with acid ethanol was 9020μU/ml, and C-peptide immunoreactivity (CPR) was 36 ng/ml in serum obtained during the attack of February 14, 1975. Major portions of the IRI and CPR were extracted from serum IgG which was precipitated with anti-human IgG antiserum.
By radioimmunoelectrophoresis, specific precipitation method and Christiansen's method, insulinbinding antibodies of the patient were found only in IgG. Light chains of the insulinbinding globulins were mainly of the kappa type, as compared with diabetics treated with insulin in whom the lambda type could not be disregarded besides the kappa type. The patient's serum showed only non-specific binding to, ¹²⁵I-porcine proinsulin, although diabetics treated with insulin had specific antibodies to porcine proinsulin in the serum.
The diagnosis of insulin autoimmune syndrome was made on this patient who showed rapid and spontaneous improvement after the attacks of spontanous hypoglycemia.

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A 25 year old male patient, who had suffered from diabetes mellitus for 5 years developed systemic allergy to insulin during lente insulin treatment. He had generalized erythema and vesicle formation and low blood pressure. After discontinuation of insulin treatment, the patient developed diabetic ketoacidosis which was treated by requent intramuscular injections of low doses of insulin. The patient was also administered steroid and antihistamine preparations for generalized insulin skin allergy. Diphenhydramine hydrochloride appeared to be effective for the insulin allergy.
Intradermal tests with commercially available insulin preparations including monocomponent semilente insulin showed strongly positive skin reactions. However, desensitization by monocomponent insulin preparation was attempted. A rapid desensitization procedure on the first day ended in failure. Subsequently, another slower desensitization schedule was begun on the second day. Allergic reactions from intradermal tests were observed on the second and third day, but no reactions were noted from subcutaneous injections. On the seventh day, the patient was given 2 units of insulin from subcutaneous injection at one time without any undesirable reactions and insulin dosage up to 20 units per day on the 22nd day.: Thereafter, he was treated by monocomponent lente insulin and was in a well-contrlled condition. The IgE level of the patient, which had been markedly elevated upon appearance of insulin allergy, was decreased toa nearly normal level by treatment with monocomponent insulin.

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Plasma membranes of pig liver were isolated by means of an aqueous two-phase polymer system and sucrose gradient. The plasma membranes were demonstrated to have high purity when measured by 5'-nucleotidase activity.
To our knowledge, this is the first demonstration of radioreceptor assay for insulin using pig liver plasma membranes. The technical performance of the assay is easy and rapid, and the assay system has a detection limit of 15μU/m/ of porcine insulin.
A radioreceptor assay for insulin should provide a useful tool for evaluation of biological activity of circulating human insulin and its derivatives.

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Conventional insulin preparations (Novo), 5 times recrystallized, were contaminated with approximately 1 to 2 % proinsulin-like substances. Anti-insulin antibodies and even proinsulin-specific antibodies were found in many cases of diabetics treated with these insulin preparations. Monocomponent insulin, purified in order to eliminate its antigenicity for insulin, appeared to be less immunogenic than conventional insulin.
In this study, the contaminating proinsulin-like substances in Monocomponent Lente insulin (Batch 522*5), Monocomponent Actrapid insulin (Batch 500*5) and conventional Lente insulin (Lot. No.297961) were estimated directly and after gel filtration by porcine proinsulin-speufic specific radioimmunoassay.
Although gel filtration of conventional insulin (500 ng) revealed proinsulin-like components (PLC), these PLC were not detectable after gel filtration of 500 ng and 5000 ng of Monocomponent insulin by specific radioimmunoassay.
Monocomponent insulin preparations contained less than O.0001% of porcine proinsulin-like immunoreactivity (PLI) by weight as estimated directly utilizing specific radioimmunoassay for porcine PLI.

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An association has been demonstrated between the HLA antigens and Bw 15 and juvenile dibetes mellitus in Caucasians. However HLA-B 8 is almost absent in the Japanese. For this reason, a racial point, HLA typing was performed by a lymphocytotoxicity micromethod on 60 insulin-dependent diabetes mellitus patients who had developed diabetes mellitus by the age of 15 years, and on 355 healthy controls.
27 HLA antigens were tested with 100 selected antisera.
In these patients, we observed an increased incidence of HLA-BW 22.1: 25.0 per cent in patients as against 5.6 per cent in controls, HLA-BW 22.2: 23.3 per cent in patients 4.5 per cent in controls. This difference is highly significant.

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