David L. Horwitz

Determination of free and total insulin and C-peptide in insulin-treated diabetics.

Serum levels of free and total insulin as well as total C-peptide immunoreactivity (C-peptide and proinsulin) and C-peptide were measured in insulin-treated diabetics with circulating insulin antibodies by the addition of polyethylene glycol (PEG) before and after acidification. PEG resulted in complete precipitation of insulin antibodies from serum and made it possible to measure free insulin in the supernatant. Incubation of serum at 37δ C. for two hours before addition of PEG resulted in values for free insulin that probably resembled the in-vivo levels most closely. The same method could also be used to remove proinsulin bound to circulating insulin antibodies and permitted the measurement of C-peptide in the supernatant. Clinical studies using this approach indicate that combined measurements of serum free and total insulin and C-peptide provide information that is helpful in understanding the contribution of endogenous and exogenous insulin to the course and metabolic control of insulin-requiring diabetic patients.

Proinsulin, insulin, and C-peptide concentrations in human portal and peripheral blood.

Concentrations of insulin, proinsulin, and C-peptide were measured in portal and peripheral venous blood in six nondiabetic, nonobese subjects. Portal vein samples were obtained by umbilical vein catheterization. Three subjects were studied with intravenous infusion of 25 g glucose, and three with 30 g arginine. Insulin and proinsulin were determined in the insulin immunoassay after separation by gel filtration, and C-peptide was measured by direct immunoassay. With both glucose and arginine stimulation, portal vein levels of all three peptides peaked at 90-120 s after the onset of the stimulus. Relative increases in insulin concentration were greater than those of proinsulin or C-peptide. In peripheral venous blood, maximal levels of the three peptides were observed later (2-5 min), and the increase in insulin relative toproinsulin and C-peptide was not as great. At the time of peak secretion, portal vein insulin and C-peptide approached equimolar concentrations, and proinsulin, as measured against an insulin standard, comprised approximately 2.5% of the total immunoreactive insulin. After stimulation by glucose or arginine, portal insulin, proinsulin and C-peptide levels were not correlated with the concentrations measured in simultaneously drawn peripheral samples. At all sampling times, however, significant correlation was found between insulin and C-peptide in both peripheral and portal blood. The results indicate that under the conditions studied, insulin and C-peptide are secreted in equimolar concentrations in man, and that proinsulin is secreted in the same proportion to insulin as found in the pancreas. Consideration of the relative secretory and metabolic rates of the three beta cell peptides explains their peripheral concentrations. The data further support the use of plasma C-peptide as an indicator of beta cell secretory 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 antigenk 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 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.

Kinetics of human connecting peptide in normal and diabetic subjects.

The metabolic clearance rate (MCR) of synthetic human connecting peptide (C-peptide) was measured with a single-dose injection technique in six normal and seven diabetic subjects and with a constant infusion technique in one normal subject. The MCR of C-peptide did not differ in normal subjects (4.4 ml/min per kg; range, 3.7-4.9) and in diabetic subjects (4.7 ml/min per kg; range, 3.7-5.8). Employment of both techniques in one subject gave similar MCR. The average half-life of C-peptide in plasma calculated from the last 1-h period of the single-dose injection studies was longer in the insulin-dependent diabetics (42.5 min; range, 39.4-48.5) than in the normal subjects (33.5 min; range, 24.9-45.3). These results indicate that the beta-cell secretory capacity of normal and insulin-dependent diabetic subjects can be compared by measuring the C-peptide concentration in peripheral venous plasma. The difference in the half-life of C-peptide in plasma between diabetics and normals suggests an altered kinetics of the disappearance of the peptide, while the overall metabolism, as expressed by the MCR, is similar.

High-fiber diets in the treatment of diabetes mellitus.

Eight patients with insulin-requiring diabetes were given, in random order, a diet containing 3 g and one containing 20 g of crude fiber. Each diet was maintained for 10 days and was the same in calories, carbohydrate, fat, and protein. Insulin dose was kept constant. Mean plasma glucose on the low-fiber diet was 169.4 +/- 11.7 mg/dl, significantly higher (P less than 0.001) than the value of 120.8 +/- 10.1 on the high-fiber diet. Hypoglycemic reactions were more common on the high-fiber diet. Weight remained essentially constant on both diets. Mean serum glucagon level on the high-fiber diet was significantly lower (P less than 0.001) than on the low-fiber diet, while serum free insulin levels were unchanged. These data indicate that substantial changes in fiber content of the diabetic diet may lead to marked changes in diabetic control and that increasing dietary fiber may be a useful means of lowering plasma glucose in some diabetic patients.

Factitious hypoglycemia. Diagnosis by measurement of serum C-peptide immunoreactivity and insulin-binding antibodies.

In seven patients with factitious hypoglycemia due to the surreptitious injection of insulin, we made the diagnosis by measurements of plasma insulin and C-peptide immunoreactivity (in seven patients), facilitated by the finding of circulating insulin-binding antibodies (in two patients). The simultaneous demonstration of low plasma glucose, high immunoreactive insulin and suppressed C-peptide immunoreactivity represents a triad of results pathognomonic of exogenous insulin administration. Determination of plasma free C-peptide and free insulin permitted patients with high titers of insulin antibodies, including those with a history of insulin-treated diabetes, to be studied and diagnosed in a way similar to that in subjects who had no circulating insulin antibodies.

Inhibition of Insulin Secretion by Exogenous Insulin in Normal Man as Demonstrated by C-peptide Assay

To investigate whether insulin exerts feedback regulation of its own secretion, paired studies were performed in normal men in two separate protocols. In the first protocol, four men were studied on two occasions. On one occasion, insulin was infused at 5 μU. per kilogram per minute for 120 minutes, achieving arterial insulin levels of 600 to 700 μU./ml. With use of a variable glucose infusion, the plasma glucose concentration was maintained at fasting levels for 60 minutes and then raised abruptly to 165 mg./dl. and was maintained at that level for the remaining 60 minutes. On a second occasion, the study was repeated except that saline was infused instead of insulin. The plasma glucose concentration remained at fasting levels until the last 60 minutes, when it was similarly raised to 165 mg./dl. and maintained at that level until the end of the study. Connecting peptide reactivity (CPR) was measured as an index of endogenous insulin secretion in the presence of exogenous insulin. During the initial 60 minutes of the study, with euglycemia maintained, there was a significant (46 per cent) decline (p < 0.01) in the levels of CPR in the insulin-treated subjects. At 60 minutes, when hyperglycemia was induced, CPR rose in both groups. The rise of CPR in the insulin-treated group, however, was significantlyless (p < 0.01) than in the saline control group. To investigate whether insulin inhibition of insulin secretion during hyperglycemia would occur without prolonged insulin pretreatment, paired studies were performed in three additional men. In this protocol, insulin (5 mU. per kilogram per minute) or saline was infused for 70 minutes. Euglycemia was maintained for just 10 minutes. Thereafter, the plasma glucose concentration was. raised to 170 mg./dl. in both groups. This acute induction of hyperglycemia without prolonged insulin pretreatment resulted in similar increases in CPR in both insulin- and saline-treated groups. From these data we conclude that (1) exogenous insulin administration, with maintenance of euglycemia, results in significant inhibition of basal insulin secretion; (2) the administration of exogenous insulin for 60 minutes before and for 60 minutes after the acute induction of hyperglycemia results in significant inhibition of glucose-stimulated insulin secretion; and (3) exogenous insulin administered for just 10 minutes before and during the acute induction of hyperglycemia, however, does not result in inhibition of the insulin response to the hyperglycemic stimulus.

Quantitation of Human Pancreatic Beta-cell Function by Immunoassay of C-Peptide in Urine

Human proinsulin connecting peptide (C-peptide) was measured by immunoassay in urine from 25 normal subjects, 18 patients with diabetes mellitus, and 34 patients with various degrees of renal insufficiency. Assay validation studies showed that pancreatic C-peptide was quantitatively recovered when added to urine. Fractionation of urine by gel filtration indicated that most endogenous C-peptide eluted in fractions that corresponded to the C-peptide standard. In 34 nondiabetic subjects with normal kidney function or various renal diseases, C-peptide clearance was independent of creatinine clearance over a range of 6 to 190 ml./min. Urine C-peptide clearance (5.1 ± 0.6 ml./min.) is greater than that of insulin (1.1 ± 0.2 ml./min.), and the total quantity of C-peptide excreted in the urine per day represents 5 per cent of pancreatic secretion, as against only 0.1 per cent of secreted insulin. Healthy subjects excreted 36 ± 4 μg. C-peptide per 24 hours, while this value in juvenile-onset diabetics was only 1.1 ± 0.5 μg. Adult-onset diabetics excreted 24 ± 7 μg./24 hr., the range overlapping the excretory rates of both normal subjects and juvenileonset diabetics. Two insulin-requiring adult-onset diabetics showed significant beta-cell reserve during the course of acute infections. These results suggest that urine C-peptide provides a useful means of assessing beta-cell secretory capacity over a period of time and is especially advantageous when frequent blood sampling is not feasible.

Control of juvenile diabetes mellitus and its relationship to endogenous insulin secretion as measured by C-peptide immunoreactivity

Proinsulin is converted to insulin and C-peptide in the pancreatic beta-cells; the latter two peptides are secreted in equimolar concentrations. Thus measurements of C-peptide immunoreactivity may provide a means of assessing residual pancreatic function in insulin-treated diabetic patients. Thirty-five patients with a mean (+/- SE) age of 13.4 +/- .6 years who had diabetes mellitus for 4.8 +/- .3 years were included in this study. Glucose and CPR were measured in the fasting state and one hour after 1 gm/kg (maximum 50 gm) of oral and glucose. Patients were assigned to one of two groups on the basis of adequate or poor control of diabetes. Twenty-five of the 35 (71%) patients had evidence of endogenous beta-cell function, i.e., CPR greater than 0.5 ng/ml. CPR levels over 0.5 ng/ml were present in a significantly (p less than 0.05) greater number of patients with diabetes of less than 5 years duration (19/21) than in those with diabetes greater than 5 years duration (6/14). Only one patient showed a rise in CPR after the glucose load. All patients with CPR greater than 2.0 ng/ml were in the adequately controlled groups, but there were patients with CPR less than 2.0 ng/ml in both adequately and poorly controlled groups. Because the CPR value includes both C-peptide and antibody-bound proinsulin, separate determination of free C-peptide was done in 30 patients. These results confirmed the conclusions based on CPR estimation. Although growth hormone values were higher in patients in the poorly controlled group, there was no correlation between hGH and CPR. We conclude that residual insulin secretion in diabetic patients may facilitate good control, but that low CPR values and hence absent beta-cell reserve is not always associated with poor control.

Insulin and the Kidney

Changes in renal function and structure are frequently observed in patients with diabetes mellitus. In the early phases of the disease, alterations in glomerular filtration rate, renal plasma flow, glomerular permeability and tubular capacity for glucose reabsorption occur. In the late stages of juvenile onset diabetes, renal failure is a common cause of death. For this reason, increasing attention is being paid to the possibility of long-term dialysis and renal transplantation in these patients. The kidneys play an important role in regulating insulin metabolism. The renal arteriovenous difference is approximately 30-45% and a linear relationship exists between the arterial insulin level and the renal arteriovenous concentration difference. The renal extraction of insulin is 200 ml/min in man, and it is estimated that 6-8 U are removed and degraded by the kidney in 24 h. The quantity of insulin in urine is small. However, its clearance is relatively constant over a wide range of serum concentrations and is 0.15-0.5 ml/min. The mean basal insulin excretion is 3.6 muU/mg creatinine, and a fourfold rise occurs following a glucose load. The urinary insulin values in neonates, children and patients with diabetes and renal failure are reviewed. In diabetic patients, progressive renal disease is accompanied by decreasing insulin requirements. In contrast, nondiabetic patients who develop renal failure frequently show abnormalities in carbohydrate metabolism, the commonest of which is a pseudodiabetic state.