beta-cell function in children with diabetes.

Although it is generally believed that insulin secretion is minimal or absent in juvenile-onset diabetes, we have found appreciable levels of C-peptide at the time of onset in 12 patients, 4 to 16 years old (9.3 +/- 4.2). Ten of them had ketonuria but none severe ketoacidosis. All entered a remission period. Most of the patients had near normal C-peptide levels during the remission, and their beta cells had the capacity to respond to a breakfast stimulation with increased insulin secretion. C-peptide and proinsulin were also determined in 98 juvenile diabetics with age at onset of 1 to 16 years (6.8 +/- 3.9) and a duration of diabetes between two and 17 years (6.7 +/- 3.4). Many were found to have persisting beta-cell function, which seems to be of importance for ensuring stability in metabolic control. Although little is known about factors that may slow or reverse the process leading to beta-cell failure, our results suggest that early detection and intensive treatment of diabetes before severe metabolic disturbances have occurred may help preserve beta-cell function.

Characterization of seven C-peptide antisera.

The plasma C-peptide immunoreactivity (CPR) in 10 normal subjects varied considerably when measured with different antisera in parallel assays. The CPR level correlated with the blank "CPR" value measured in plasma devoid of C-peptide and to a lesser degree with the sensitivity of the standard curves obtained with the individual antisera. Storage of plasma samples at different temperatures and for different lengths of time before the analyses were carried out resulted in further variation in the CPR results. This was caused by a time- and temperature-dependent fall in CPR, which was more pronounced with some antisera than with others. This sensitivity to storage of plasma did not correlate with the antigenic characteristics of the antisera as determined by their reactivity with 11 specific fragments of the C-peptide molecule. The contribution of human proinsulin to the CPR concentration relative in normal subjects was considered to be negligible even though the relative immunoreactivity of human proinsulin and C-peptide ranged from 11 to 143 per cent among these antisera. These results suggest that differences in C-peptide antisera are a major reason for the variation in the concentration of circulating CPR as measured in different C-peptide immunoassays.

The long-term effects of chlorpropamide on insulin, C-peptide, and proinsulin secretion.

It had been suggested that long-term lowering of blood glucose by sulfonylureas in non-insulin-dependent (NIDD) diabetes is not due to a sustained increase in insulin secretion. We have re-examined this question. Thirteen nonobese NIDD patients not controlled on diet alone were studied prospectively on treatment with chlorpropamide for over 3 mo. Of these, 9 were also studied after 1 yr. Improvement in glucose tolerance was associated with an increase in fasting and postglucose serum insulin and C-peptide concentration. We conclude that at least for over 1 yr chlorpropamide increases insulin secretion. After 3 and 12 mo the fasting proinsulin percentage of immunoreactive insulin was increased.

Insulin-specific IgE in serum of 67 diabetic patients against human insulin (Novo), porcine insulin, and bovine insulin. Four case reports.

We have investigated the binding of insulin-specific IgE (IgE1) to porcine, bovine, and human insulin (Novo), pancreatic polypeptide, and a-component in serum samples from type I diabetic patients treated with insulin preparations of different purity. Patients treated with porcine or mixed-species purified insulin (monocomponent) did not differ significantly from a nondiabetic control group. Hence, in these groups no IgE1 could be detected against any of the components investigated. Serum samples from patients treated with five-times recrystallized insulin preparations and patients with insulin allergy showed a significantly greater binding of IgE1 to the three species of insulin; IgE1 binding was greatest to bovine insulin and least to human insulin. The difference in binding was most significant in the allergic patients (P less than 0.001), probably due to differences in the affinity. It is concluded that conventional (recrystallized) insulin induces more IgE1 than monocomponent insulin. Data are presented on confirmed allergy in four diabetic patients whose allergy disappeared upon transfer to human insulin.

The clinical pharmacology of human insulin (Novo) in normal subjects.

Neutral regular human insulin (Novo) (derived from porcine insulin) and porcine insulin were administered by subcutaneous (s.c.) injection into the anterior abdominal wall in two different groups of six normal male subjects. The insulin dosage was 0.075 IU/kg body wt; diluting medium was used to obtain control values. In one group, somatostatin was administered by continuous intravenous infusion (100 micrograms/h) to inhibit pancreatic beta-cell secretion. The plasma glucose, C-peptide, and insulin (immunoreactive) responses to human insulin and porcine insulin were identical in the studies with and without somatostatin. Although the incremental plasma insulin values achieved with the two insulins were similar in the two studies, the hypoglycemic effect was accentuated in the presence of somatostatin, with a delayed recovery toward normoglycemia. The human insulin and porcine insulin were well tolerated in all subjects and there were no unwanted side effects.

Differential changes in the 5-hydroxytryptamine and insulin content of guinea-pig B-cells.

In the guinea-pig pancreas, 5-hydroxy-tryptamine (5-HT) occurs in the B-cells as well as in enterochromaffin cells scattered in the exocrine parenchyma. In the present study we examined the effects on the pancreatic 5-HT and insulin content of drugs known to affect the B-cells. For this purpose a radioimmunoassay of guinea-pig insulin was developed. Streptozotocin reduced the number of detectable B-cells and partially degranulated those that remained. It also lowered the insulin content of the pancreas and the 5-HT content of the B-cells but did not affect the 5-HT content of the enterochromaffin cells. Reserpine lowered the 5-HT content of both B-cells and enterochromaffin cells. The number and ultrastructure of the B-cells and the pancreatic insulin content was not affected. Streptozotocin + reserpine seemed to have additive effects of B-cell 5-HT. The results with these 2 drugs indicate that roughly 50% of pancreatic 5-HT is contained in the B-cells. Diazoxide markedly increased the pancreatic insulin content without affecting pancreatic 5-HT. Despite the fact that 5-HT and insulin are stored together in the cytoplasmic granules of the B-cells, 5-HT was differentially depleted from this store by reserpine. A marked disproportionality between 5-HT and insulin content was also induced by diazoxide.

Structure-function relationship: immunologic.

It is suggested that the antigenic site of glucagon for the specific sera is located within the 24-29 section of the molecule and within the 2-23 section for the fully cross-reacting sera. Biologically inactivated glucagon may retain immunoreactivity in spite of the loss of receptor-binding activity.

Influence of portal delivery of insulin on intracellular glucose and lipid metabolism.

We have investigated whether portal delivery of insulin as a result of intrahepatic islet cell autografts would prevent the development of metabolic alterations. Seven pancreatectomized dogs received islet autografts transplanted into the liver through the portal vein (PD). One year after transplantation, their intravenous glucose tolerance and insulin responses were similar to age-matched control (C) dogs (n = 5). Also, normal triglyceride content in arterial smooth muscle and striated muscle was observed in the dogs with portal insulin delivery in contrast to the substantial increases we observed in pancreatectomized dogs (n = 7) with pancreatic autografts that drained into the systemic circulation (SD). In these dogs, the tissue samples were taken at the age of 3 to 4 years. Triglyceride content (mean +/- SEM) in the aorta was 4.9 +/- 1.2 versus 2.6 +/- 0.6 versus 20.7 +/- 8.0 mumol/g (P less than .01) in C, PD, and SD models, respectively. The corresponding values for triglyceride content in striated muscles were 29.1 +/- 1.2, 25.9 +/- 1.5, and 171.4 +/- 46.6 mumol/g (P less than .01). Glucose-6-phosphate dehydrogenase (G-6-PDH) and malic enzyme, key enzymes for lipid synthesis, were also normal in the PD model, in contrast to the fivefold increased activity of these enzymes in the SD model (P less than .01). The glycolytic enzymes, hexokinase (HK) and phosphofructokinase (PFK), were normal compared with the decreased values in the SD. These data indicate that it is possible to normalize glucose and lipid metabolism in arterial walls by portal delivery of insulin, following intrahepatic islet cell transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Human insulin. Facts and perspectives.

Recently an improved process for the production of human insulin from porcine insulin has been described. After being characterized, this insulin has been tested and shown to be safe and effective in diabetics in a short-term, double-blind, cross-over study. The availability of sufficient quantities of this human insulin now permits short- and long-term clinical investigations of the possible benefits of human insulin in the treatment of diabetics.

Radioimmunological determination of human C-peptide in serum.

A routing radioimmunoassay for human C-peptide in serum is described. Antibodies against human C-peptide were raised by immunizing guinea pigs with human b-component. Nine out of 12 animals produced useful antibodies within 6 months. Insulin antibodies coupled to Sepharose were used to bind human proinsulin and insulin in the serum and after centrifugation C-peptide was determined in the supernatant. The detection limit of the assay (calculated as 2 SD from zero) was about 0.003 pmole of C-peptide (in 100 mul). The main sources of error were: (1) Normal and diabetic sera devoid of C-peptide gave a displacement of 125I-Tyr-C-peptide varying from 0 to 0.16 nM (6 different antisera). Only one antiserum (M 1181) showed no displacement, and the values of C-peptide determined with this antiserum in normal and diabetic sera were lower than the values determined with another antiserum, which gave a value of 0.07 nM in the sera free of C-peptide. It is suggested that displacement found with most antisera is due to substances in serum that are not related to C-peptide or proinsulin. (2) Serial dilutions of pancreatic extracts and sera may yield dilution curves slightly different to those of the synthetic standard. Possible explanations are discussed. These sources of error can be eliminated or reduced by the proper selection of antisera. Fasting sera from 15 normals, 8 maturity-onset diabetics and 10 insulin-requiring diabetics showed the following concentrations of C-peptide: (M 1181) 0.35 +/- 0.09, 0.74 +/- 0.51 and 0.21 +/- 0.14 (nM, mean +/- SD). One hour after 1.75 g/kg oral glucose the values increased to 2.24 +/- 0.71, 2.34 +/- 0.24 nM.

B-cell response to exercise in diabetic and non-diabetic children.

20 non-diabetic and 11 insulin dependent diabetic (IDD) children underwent short time (20 min) bicycle ergometer exercise followed by a 10 min rest period. Glucose, IRI, C-peptide and proinsulin were determined prior to and at the end of the exercise, and again after 10 min rest. While no significant change in mean glucose was observed during exercise in the non-diabetics, significant decreases were observed in IRI, C-peptide and proinsulin. After 10 min rest glucose as well as the three B-cell secretory products increased significantly. The change in glucose was significantly (p less than 0.001) correlated to the change in IRI. In the resting period IRI rose more than C-peptide in some subjects. IRI even rose without simultaneous rise in C-peptide indicating a release of tissue bound IRI. The group of IDD children did not show any significant changes in glucose and total IRI, while the endogenous insulin, as measured by C-peptide, did show a fall during exercise. The same was found for proinsulin. The lack of increased endogenous secretion during the rest period was most likely due to suppression of B-cell due to hyperinsulinism and lack of increased glucose concentrations during the rest period.

Separation of the two double-chain bovine intermediates of the proinsulin-insulin conversion I. Chemical, immunochemical, circular dichroism, and biological characterization.

The intermediates of the proinsulin-insulin conversion were separated by cation exchange. The circular dichroism spectra of the intermediates showed less alpha-helix than insulin and proinsulin. It is suggested that the C-peptide interacts with the section of alpha-helix contained between residues 2 and 8 in the A-chain of the insulin moieties and unwinds the alpha-helix. The in vivo activities of the intermediates were found to be in the order of 50% relative to insulin. In the fat cell assay, the A-chain-substituted form is weaker (9%) than the B-chain-substituted form (19%). The C-peptide segments of the two forms reacted with C-peptide-specific antibodies as fully as the free C-peptide, on a molar basis. In contrast, the insulin segments were hindered from reacting with insulin-specific antibodies as fully as the insulin.

Human C-peptide in normal and diabetic subjects.

Concentrations of human C-peptide, IRI (immunoreactive insulin) and glucose were determined during oral glucose tolerance test (1.75 g glucose/kg ideal body weight) in 14 normal persons (N), 9 maturity-onset diabetics (DI) and 10 insulin-requiring diabetics (DII) never treated with insulin and in 3 formerly insulin treated diabetics. The mean fasting levels of C-peptide and IRI in the first three groups were: N: 0.37 +/- 0.02 nM and 0.048 +/- 0.009 nM, DI: 0.86 +/- 0.17 nM and 0.11 +/- 0.029 nM, DH: 0.37 +/- 0.04 nM and 0.063 +/- 0.009 nM. One hour after oral glucose ingestion, the respective values increased to: N: 2.53 +/- 0.20 nM and 0.52 +/- 0.077 nM, DI: 2.49 +/- 0.31 nM and 0.49 +/- 0.11 nM, DH: 0.49 +/- 0.05 nM and 0.11 +/- 0.014 nM. Although secreted from the pancreas in equimolar concentrations, the molar ratio of C-peptide to insulin in peripheral blood was about 7 in the fasting state, falling to about 5 in the glucose stimulated condition. Maturity-onset diabetics had higher fasting levels of C-peptide than normal subjects, in agreement with the IRI levels. Three patients previously treated with insulin and having insulin antibodies showed C-peptide responses significantly below the normal range. In one of these patients, the test was repeated 9 months later when the insulin antibodies had disappeared, and the C-peptide response observed at that time was much higher. It is suggested that insulin antibodies cause an impaired IRI - and consequently C-peptide response - by constantly removing insulin from the granules in the B-cell. In normal humans the peripheral C-peptide responses to the oral glucose load showed less relative variation than do the insulin responses. Therefore, a radioimmunoassay for C-peptide in addition to the assay for insulin will provide supplementary information on insulinsecretion.

Hypersecretion of proinsulin in thyrotoxicosis.

The plasma insulin, C-peptide and proinsulin concentrations were investigated in thyrotoxic patients and in normal controls after an overnight fast, during a 36 h fasting period, an intravenous glucose tolerance test and an oral glucose tolerance test. The main finding was a significantly raised concentration of proinsulin in plasma of patients with thyrotoxicosis. After an overnight fast the plasma proinsulin was 0.048 +/- 0.005 pmol/ml in 15 thyrotoxic patients compared with 0.023 +/- 0.012 pmol/ml in 15 euthyroid controls. A twofold rise of plasma proinsulin concentration was also found in thyrotoxic patients during a prolonged fast, and during intravenous and oral glucose tolerance tests. The immunoreactivity of proinsulin in the insulin radioimmunoassay gave rise to slightly elevated concentrations of immunoreactive insulin in thyrotoxic patients in all the conditions investigated. When insulin values were corrected for proinsulin crossreactivity, they were similar in euthyroid controls and thyrotoxic patients. The concentration of plasma C-peptide was not significantly altered in thyrotoxic patients during intravenous and oral glucose tolerance tests.

Increased release of gut glucagon in reactive hypoglycaemia.

A patient with reactive hypoglycaemia showed hypersecretion of insulin in response to enteral beta-cell stimulation. Increased levels of gut glucagon-like immunoreactivity in serum were demonstrated by use of specific antisera. Other measurable hormones influencing carbohydrate metabolism were within normal limits. It is suggested that gut glucagon-like immunoreactivity may be of pathogenetic significance in reactive hypoglycaemia.

C-peptide in juvenile diabetes.

C-peptide can be used as a measure of endogenous insulin secretion in insulin treated diabetics with insulin antibodies. At the onset of juvenile diabetes insulin production is thought to be absent or minimal, but we have found rather high levels of C-peptide, even in ketoacidotic patients. The ketoacidosis does not mean an irreversible beta cell failure. In the postinitial remission period with stable metabolism many patients have normal or almost normal C-peptide levels and their beta cells have the capacity to respond to natural stimulation with an increased insulin secretion. For some unknown reason the metabolism becomes more labile coinciding with decreasing C-peptide values. However, even several years beyond the postinitial remission period many juvenile diabetics have some persistent beta cell function, and it has been shown that even trace remnants of beta cell function are of importance for stabilization of the metabolism. There is no reason to believe that the beta cell failure should be predetermined e.g. by genetic factors. However, little is known how to influence the progression and stop the increasing beta cell failure. Some of our results suggest that an early detection and an intensive treatment of diabetes before severe metabolic disturbances and pronounced insulin deficiency have appeared, may increase the possibility of preserving some beta cell function.