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We describe a family from Japan displaying the mutant insulin syndrome with hyperinsulinaemia and an increased insulin: C-peptide molar ratio. Serum insulin isolated from several family members showed reduced in vitro biological activity, and analysis by high performance liquid chromatography revealed a peak co-eluting with human insulin and a second species of increased hydrophobicity co-migrating with the previously reported Insulin Wakayama. The insulin genes from the propositus were cloned and sequenced, revealing one normal allele; the second allele, encoding a leucine for valine amino acid substitution at position 3 of the insulin A chain, was similar to that previously described for Insulin Wakayama. Synthesized [LeuA3] insulin showed 0.14% of receptor binding activity on rat adipocytes and a 10-fold prolonged half-life in a somatostatin-infused dog compared with human insulin. The finding of the same mutant gene in two unrelated Japanese families suggests that Insulin Wakayama may be discovered in additional Japanese families with hyperinsulinaemia and/or diabetes.

To examine differences among pregnant diabetic and nondiabetic subjects, serum glucose, and immunoreactivity of C-peptide, free and total insulin were measured at hourly intervals during a 24--h third trimester metabolic ward evaluation. Six normals, three mild, and four juvenile-onset type diabetics were studied. Diets were identical for all subjects. Mild diabetics differed from juvenile diabetics by having significant residual pancreatic B-cell function, as measured by C-peptide immunoreactivity. Short and intermediate acting insulins given once or twice daily to diabetics maintained serum glucose levels within the normal range throughout the 24 h. Despite wide variation in serum total insulin levels, peripheral free insulin concentrations in well-controlled diabetics fell within a relatively narrow range that was higher than in controls. Infants of the diabetic subjects were comparable to the offpsring of the control women.

Rabbits were immunised with suspensions of viable, insulin-producing islet cells prepared from collagenase-isolated rat or ob/ob mouse pancreatic islets. Antibodies reactive with the surface of dispersed rat islet cells were present in both the rabbit anti-rat and the rabbit anti -ob/ob mouse islet sera as revealed by indirect immunofluorescence or by a radioligandassay using 125I-Protein A as a measure of cell bound IgG. In a competition assay the binding of 125I-Protein A was displaced in a concentration dependent manner by non-radioactive Protein A. Maximal displacement was found at concentrations of Protein A higher than 0.1 μg. added to 105 islet cells. Although not always detected by immunofluorescence there was a several-fold increase above normal rabbit serum of 125I-Protein A-binding to rat hepatocytes and spleen lymphocytes incubated with the islet cell antisera. Conversely, rabbit antisera against rat spleen lymphocytes or against a rat liver plasma membrane preparation reacted with rat islet cells. The rabbit anti-rat islet cell antiserum was absorbed to both spleen lymphocytes and hepatocytes until there was no binding of 125I-Protein A to either cell type. Islet specific antibodies were still present since this doubly absorbed antiserum induced cell surface immunofluorescence as well as 125I-Protein A-binding to rat islet cells. It is concluded that apart from common antigenic determinants immunisation with viable islet cells induces formation of antibodies directed against specific islet cell surface components.

1.Details are given of a double antibody immunoassay method suitable for measuring insulin in urine. — 2. The method proved specific and reproducible. Human insulin added to urine was quantitatively recovered and dilution and concentration resulted in proportional changes in measured insulin. — 3.Urines kept at 4°C for 24 hours showed no loss of assayable insulin, while decreases of 4–23 % were observed after standing at room temperature for 24 hours; samples of urine adjusted to pH 7.4–8.4 and centrifuged were kept at −20°C for 9 months without loss. Addition of albumin to urine to prevent adsorption of insulin on the container was found to be unnecessary. — 4. Tests of the first antibody reaction showed that non-specific co-precipitation of radioactive insulin was less than 3.5 % and that addition of sodium chloride did not alter the standard insulin curve. Monitor tubes containing131I-guinea-pig gamma-globulin showed optimal second antibody precipitation in all the urine assays surveyed. — 5. In normal subjects, the renal clearance of insulin was relatively constant over a wide range of serum insulin levels, but varied with body size. However, preliminary studies suggested that the clearance was reduced in pregnancy, hypertension and peripheral vascular disease, all of which are characterized by high serum insulin values. In contrast, the clearance in acromegaly was normal as both serum and urine insulin levels were raised. In chronic renal disease the urinary excretion of insulin was very high. — 6. Direct measurement of urinary insulin was feasible in insulin-treated diabetics provided that renal function was normal; insulin-binding antibodies were found in the urine of these diabetics only in association with proteinuria.

Previous studies have shown a variety of immunological abnormalities in Type 1 (insulin-dependent) diabetes, including disturbances in peripheral lymphocytes and anti-lymphocyte antibodies. We measured subsets of T and natural killer cells with monoclonal antibodies in patients with diabetes, and also assayed for anti-lymphocyte antibodies using dual colour immunofluorescence and flow cytometry. We found a significant decrease in numbers of Leu 3a (helper/inducer) cells in Type 1 diabetic patients of recent onset and intermediate levels in patients with longer duration of the disease. Leu 4 (pan T cell) levels were reduced in Type 1 diabetic patients of more than 4 months duration. Leu 7 (natural killer cells) were increased in Type 2 (non-insulin-dependent) diabetic patients. Individual Type 1 diabetic patients of recent onset showed low levels of Leu 7 and 11 a (natural killer cell) levels with normal 3a levels. Autoantibodies against Leu 3a + cells were present in higher titres in the Type 1 diabetic patients of recent onset than in control subjects. We conclude: (1) Leu 3a cells may be decreased in Type 1 diabetic patients of recent onset and return to normal with time; (2) low numbers of Leu 7 and 11a cells with normal numbers of Leu 3a may be seen in some Type 1 diabetic patients of recent onset, which may help explain previous reports of decreased suppressor cells; (3) Leu 7 levels may be increased in Type 2 diabetes; (4) autoantibodies against Leu 3a + peripheral lymphocytes may be seen in Type 1 diabetic patients of recent onset. These appear to be a marker of autoimmune phenomena rather than immunological mediators.

Two patients with newly diagnosed insulin dependent diabetes mellitus were treated with human leukocyte interferon based on the hypothesis that the diabetes was induced by an active viral infection in the pancreatic islets and could be arrested. High peak levels of serum interferon were achieved (100–200 U/ml) with minimal systemic side effects. There was no sustained therapeutic benefit as measured by increased production of endogenous insulin, or of C-peptide, or by a lower requirement for exogenous insulin. Further trials with interferon treatment should be undertaken only if evidence of active viral infection (culture, antigen detection) can be associated with insulin dependent diabetes onset and these markers followed during treatment.

Two new batches of pancreatic human proinsulin have been compared with biosynthetic human proinsulin. Standards of these three proinsulin preparations were made on the basis of quantitative amino-acid analyses and compared in two proinsulin radioimmunoassays with a proinsulin standard prepared 14 years ago. The curves of the new standards were superimposable. However, they differed considerably from the curve of the old standard which proved to be only one-third of the strength of the new standards, thereby leading to a threefold over-estimation of proinsulin concentrations when the old standard is used. We conclude that the new standards should replace previously used standards.

An artificial beta cell has been used to achieve and maintain a preset plasma glucose concentration in five diabetic patients undergoing surgery. These subjects were compared to control groups of normal subjects receiving either saline or glucose, and diabetics receiving glucose intraoperatively. Hyperglycaemia during surgery was seen in normals (mean plasma glucose +/- SEM: 185 +/- 16 mg/dl) and, to a greater degree, diabetics (247 +/- 36 mg/dl) receiving glucose. Insulin and C-peptide levels did not increase during 2 hours of operation in any of the control groups, suggesting beta cell suppression during surgery. As C-peptide levels declined similarly in normal subjects whether they received saline or glucose, the hyperglycaemia seems to be due to an inability to use exogenous glucose. This is confirmed by a correlation of maximal plasma glucose to glucose infusion rate (r = 0.78, p less than 0.01). The artificial beta cell was able to achieve the same plasma glucose after 2 hours of operation (128 +/- 21 mg/dl) as normal subjects receiving saline (110 +/- 7 mg/dl). The artificial beta cell proved to be a safe, convenient and effective way of monitoring and controlling the hyperglycaemia seen in diabetic patients undergoing surgery.

Proinsulin-like components (PLC) and insulin have been measured in 24 hr urine samples from 8 healthy subjects. The mean excretion of PLC was 45.8 ng and that of insulin 314 ng; the PLC: insulin ratio was 0.14. Urinary PLC was increased 3.5 fold in a patient with a pancreatic islet cell tumor and the PLC: insulin ratio was 0.35. The urinary PLC: insulin ratio is lower than that of serum, presumably because of the relatively lower urinary clearance of the larger molecular weight PLC.

HbA1c is the product of a reaction between glucose and the N-terminal valine of adult haemoglobin (HbA). The levels of HbA1c are increased in diabetic patients, and reflect their metabolic control. Thus, measurements of HbA1c have proved to be useful in the follow up and treatment of diabetic patients. HbA1c may prove to be valuable for assessing the relationship between diabetic control and long-term complications, as well as in studying the potential glycosylation of proteins in various organs which may occur in the diabetic state.

To examine the possibility that the concentration of circulating proinsulin-like material (PLM) might be helpful in evaluating the therapeutic response of patients with islet cell tumours, serum levels of PLM in three patients with islet cell tumours were correlated with hypoglycaemic symptoms and plasma glucose concentrations before and after treatment. In two patients ranges of fasting PLM concentration were 0.21–0.29 and 0.91–0.93 ng/ml, respectively, before treatment. After surgical excision of their islet cell adenomas, PLM concentrations decreased to 0.06–0.09 and 0.03–0.05 ng/ml. Insulin concentrations were low preoperatively in both patients and were unchanged postoperatively. The resulting relief from hypoglycaemia was paralleled by a reduction of PLM, with no significant change in insulin. In a third patient, treatment with streptozotocin resulted in marked symptomatic improvement, a 65% reduction in PLM concentration, but no significant change in insulin levels. Relapse was associated with increasing frequency of hypoglycaemic symptoms and increasing PLM concentrations. These findings suggest that changes in the levels of serum PLM may prove to be a sensitive indicator of the response of islet cell tumours to therapy.

To assess the risk and possible mechanisms of hypoglycaemia during moderate exercise in Type I (insulin-dependent) diabetic patients receiving constant insulin infusion, five insulin-dependent male diabetic patients were exercised 18 h after their last meal and 30 h after their last injection of intermediate acting insulin. Intravenous insulin was initially delivered via a closed-loop infusion system programmed to lower mean blood glucose from 11.3 +/- 1.8 to 4.8 +/- 0.4 mmol/l over approximately 3.5 h. Blood glucose was then maintained at this level for 4 h. At this time, the closed-loop infusion was discontinued and replaced by an open-loop system. The average amount of insulin infused per min during the 4 h normoglycaemic closed-loop period was calculated and this amount was infused at a constant rate during both a 30 min period of exercise on a bicycle ergometer (approximately 65% maximum oxygen uptake) and a 30 min rest period which followed. Five nondiabetic males served as control subjects. Despite significantly higher free insulin concentrations (p less than 0.05) and identical pre-exercise blood glucose concentrations, blood glucose rose during exercise only in the diabetic group (0.5 +/- 0.2 mmol/l; p less than 0.01). Changes in the serum concentrations of lactate, glycerol, glucagon, cortisol, non-esterified fatty acids and growth hormone were similar in the two groups and did not account for the increment of blood glucose in the diabetic patients. Beta-hydroxybutyrate concentrations were, however, higher in the diabetic patients at the onset of exercise (p less than 0.01) and decreased significantly more than the control subjects during exercise. We conclude that exercise under these conditions in diabetic patients is not attended by hypoglycaemia.

In 6 patients undergoing cardiac catheterization (in whom shunts were excluded) simultaneously sampled central venous and arterial blood specimens were analyzed for insulin, free fatty acids and glucose. Insulin levels were usually (4/6) 15–19% lower in arterial than central venous blood, but once was only 10% lower and once 8% higher. No consistent differences in central venous and arterial blood were noted for either free fatty acids or glucose.