To determine whether non-insulin-dependent diabetes is associated with specific alterations in the pattern of insulin secretion, we studied 16 patients with untreated diabetes and 14 matched controls. The rates of insulin secretion were calculated from measurements of peripheral C-peptide in blood samples taken at 15- to 20-minute intervals during a 24-hour period in which the subjects ate three mixed meals. Incremental responses of insulin secretion to meals were significantly lower in the diabetic patients (P less than 0.005), and the increases and decreases in insulin secretion after meals were more sluggish. These disruptions in secretory response were more marked after dinner than after breakfast, and a clear secretory response to dinner often could not be identified. Both the control and diabetic subjects secreted insulin in a series of discrete pulses. In the controls, a total of seven to eight pulses were identified in the period from 9 a.m. to 11 p.m., including the three post-meal periods (an average frequency of one pulse per 105 to 120 minutes), and two to four pulses were identified in the remaining 10 hours. The number of pulses in the patients and controls did not differ significantly. However, in the patients, the pulses after meals had a smaller amplitude (P less than 0.03) and were less frequently concomitant with a glucose pulse (54.7 +/- 4.9 vs. 82.2 +/- 5.0, P less than 0.001). Pulses also appeared less regularly in the patients. During glucose clamping to produce hyperglycemia (glucose level, 16.7 mmol per liter [300 mg per deciliter]), the diabetic subjects secreted, on the average, 70 percent less insulin than matched controls (P less than 0.001). These data suggest that profound alterations in the amount and temporal organization of stimulated insulin secretion may be important in the pathophysiology of beta-cell dysfunction in diabetes.

IntroductionThe secretion and hepatic extraction of insulin were compared in 14 normal volunteers and 15 obese subjects using a previously validated mathematical model of insulin secretion and rate constants for C-peptide derived from analysis of individual decay curves after intravenous bolus injections of biosynthetic human C-peptide. Insulin secretion rates were substantially higher than normal in the obese subjects after an overnight 57.2±2.8 nmol/m2 per 180 min, P < 0.001). Linear regression analysis revealed a highly significant relationship between insulin secretion and body mass index. Basal hepatic insulin extraction was not significantly Jifferent in the normal and obese subjects (53.1±3.8 vs. 51.6±4.0%). In the normal subjects, fasting insulin did not correlate with basal hepatic insulin extraction, but a significant negative correlation between fasting insulin and hepatic insulin extraction was seen in obesity (r = -0.63, P < 0.02). This finding reflected a higher extraction in the six obese subjects with fasting insulin levels within the range of the normal subjects than in the nine subjects with elevated fasting insulin concentrations (61±3 vs. 45±6%, P < 0.05).During the hyperglycemic clamp, the insulin secretion rate increased to an average maximum of 6.2-fold over baseline in the normal subjects and 5.8-fold in the obese subjects. Over the same time, the peripheral insulin concentration increased 14.1-fold over baseline in the normals and 16.6-fold over baseline in the obese, indicating a reduction in the clearance of endogenously secreted insulin. Although the fall in insulin clearance tended to be greater in the obese subjects, the differences between the two groups were not statistically significant.Thus, under basal, fasting conditions and during ingestion of a mixed diet, the hyperinsulinemia of obesity results predominantly from increased insulin secretion. In patients with more marked basal hyperinsulinemia and during intense stimulation of insulin secretion, a reduction in insulin clearance may contribute to the greater increase in peripheral insulin concen-trations that are characteristic of the obese state.

We undertook this study to examine the accuracy of plasma Cpeptide as a marker of insulin secretion. The peripheral kinetics of biosynthetic human C-peptide (BHCP) were studied in 10 normal volunteers and 7 insulin-dependent diabetic patients. Each subject received intravenous bolus injections of BHCP as well as constant and variable rate infusions. After intravenous bolus injections the metabolic clearance rate of BHCP (3.8±0.1 ml/ kg per min, mean±SEM) was not significantly different from the value obtained during its constant intravenous infusion (3.9±0.1 ml/kg per min). The metabolic clearance rate of Cpeptide measured during steady state intravenous infusions was constant over a wide concentration range.During experiments in which BHCP was infused at a variable rate, the peripheral concentration of C-peptide did not change in proportion to the infusion rate. Thus, the infusion rate of BHCP could not be calculated accurately as the product of the C-peptide concentration and metabolic clearance rate. However, the nonsteady infusion rate of BHCP could be accurately calculated from peripheral C-peptide concentrations using a two-compartment mathematical model when model parameters were derived from the C-peptide decay curve in each subject. Application of this model to predict constant infusions of C-peptide from peripheral C-peptide concentrations resulted in model generated estimates of the C-peptide infusion rate that were 101.5±3.4% and 100.4±2.8% of low and high dose rates, respectively. Estimates of the total quantity of C-peptide infused at a variable rate over 240 min based on the two-compartment model represented 104.6±2.4% of the amount actually infused. Application of this approach to clinical studies will allow the secretion rate of insulin to be estimated with considerable accuracy.The insulin secretion rate in normal subjects after an overnight fast was 89.1 pmol/min, which corresponds with a basal 24-h secretion of 18.6 U.

Monoclonal antibody-based two-site immunoradiometric assays are described for human insulin, proinsulin, 65-66 split and 32-33 split proinsulin. The detection limits of the assays lie in the range 0.8-2.5 pM. The assays for 65-66 and 32-33 split proinsulins do not distinguish between these substances and their respective C-terminal di-desamino derivatives. The assay of 65-66 split proinsulin does not cross-react with insulin, proinsulin or 32-33 split proinsulin. This material was undetectable (less than 1.0 pM) in plasma taken after an overnight fast in eight normal male subjects and the maximum individual concentration reached in plasma taken during an oral glucose tolerance test of these subjects was 3.8 pM. The proinsulin assay cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. The 32-33 split proinsulin assay cross-reacted 84 and 60% with proinsulin and 65-66 split proinsulin respectively. The insulin assay cross-reacted 5.3, 62 and 5.0% with intact proinsulin, 65-66 split proinsulin and 32-33 split proinsulin respectively. The very low concentration of 65-66 split proinsulin meant that this derivative did not interfere significantly with the specificity of the assays of proinsulin and insulin. The concentration of 32-33 split proinsulin could be calculated by subtracting the cross-reactivity of the measured proinsulin. The mean concentrations of insulin, proinsulin and 32-33 split proinsulin in eight young male subjects in the fasting state were (pM +/- S.E.M.) 20 +/- 0.3, 2.3 +/- 0.3 and 2.1 +/- 0.7 and at the maximum reached during an oral glucose tolerance test, 150 +/- 26, 9.9 +/- 1.4 and 19.7 +/- 6.0 respectively.

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The loss of these native dimer interactions weakens the hexameric LysB28ProB29-human insulin complex formed in the presence of phenolic ligands. Thus, it is hypothesized that the diffusion of the phenolic ligands from the site of injection results in the dissociation of hexamers directly to monomers, thereby maintaining the rapid time-action of the monomeric analog in spite of the hexameric conformation in therapeutic formulations.

The importance of ProB28 and LysB29 on the self-association of insulin was established by systematically truncating the C terminus of the B chain. The relationship between structure and association was further explored by making numerous amino acid replacements at B28 and B29. Association was studied by circular dichroism, size-exclusion chromatography and ultracentrifugation. Our results show that the location of a prolyl residue at B28 is critical for high-affinity self-association. Removal of ProB28 in a series of C-terminal truncated insulins, or amino acid replacement of ProB28, greatly reduced association. The largest disruption to association was achieved by replacing LysB29 with Pro and varying the amino acid at B28. Several of the analogs were predominantly monomers in solutions up to 3 mg/ml. These amino acid substitutions decreased association by primarily disrupting the formation of dimers. Such amino acid substitutions also substantially reduced the Zn-induced insulin hexamer formation. The formation of monomeric insulins through amino acid replacements was accompanied by conformational changes that may be the cause for decreased association. It is demonstrated that self-association of insulin can be drastically altered by substitution of one or two key amino acids.

We have determined the structure of a metastable disulphide isomer of human insulin. Although not observed for proinsulin folding or insulin-chain recombination, the isomer retains ordered secondary structure and a compact hydrophobic core. Comparison with native insulin reveals a global rearrangement in the orientation of A- and B-chains. One face of the protein's surface is nevertheless in common between native and non-native structures. This face contains receptor-binding determinants, rationalizing the partial biological activity of the isomer. Structures of native and non-native disulphide isomers also define alternative three-dimensional templates. Threading of insulin-like sequences provide an experimental realization of the inverse protein-folding problem.