Proinsulin Conversion Intermediates Research Articles

Time-Resolved Fluorescence Assays for Quantification of Insulin Precursors in Plasma and Serum.

In metabolic diseases such as obesity and type 2 diabetes mellitus, the conversion of proinsulin to mature insulin can be impaired. This could mean that insulin molecules with lower activity toward the insulin receptor can be released under conditions of high metabolic demand, resulting in an inadequate glucoregulatory response. The chapter describes a fluorescent monoclonal antibody-based protocol for measurement of human proinsulin and the proinsulin conversion intermediates (split proinsulins). An example assay is presented using serum from non-diabetic, normal body mass index individuals.

First direct assay for intact human proinsulin

We describe a sensitive two-site sandwich enzyme-linked immunosorbent assay for the measurement of intact human proinsulin in 100 microL of serum or plasma. The assay is based on the use of two monoclonal antibodies specific for epitopes at the C-peptide/insulin A chain junction and at the insulin B chain/C-peptide junction, respectively. Cross-reactivities with insulin, C-peptide, and the four proinsulin conversion intermediates were negligible. The detection limit in buffer was 0.2 pmol/L (3 standard deviations from zero). The working range was 0.2-100 pmol/L. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The mean recovery of added proinsulin was 103%. Dilution curves of 40 serum samples are parallel to the proinsulin calibration curve. Proinsulin concentrations in 20 fasting healthy subjects were all above the limit of detection: median (range), 2.7 pmol/L (1.1-6.9 pmol/L). Six fasting non-insulin-dependent diabetes mellitus and five insulinoma patients had proinsulin concentrations significantly higher than healthy subjects: median (range), 7.7 pmol/L (3.2-18 pmol/L) and 153 pmol/L (98-320 pmol/L), respectively.

Release of incompletely processed proinsulin is the cause of the disproportionate proinsulinemia of NIDDM.

The production of insulin from proinsulin involves cleavage of intact proinsulin into proinsulin conversion intermediates by the processing of enzymes PC2 and PC3 before fully processed insulin is produced. Intact proinsulin and these conversion intermediates are measured in many immunoreactive insulin (IRI) assays, and therefore contribute to the absolute IRI measurement. The proportion of basal IRI made up of proinsulin (PI)-like molecules (PI/IRI) is increased in NIDDM. Whether stimulated IRI levels are similarly made up of disproportionately increased PI/IRI or whether the relative proportions of proinsulin and its conversion intermediates are altered has not been evaluated. An index of the efficiency of proinsulin processing within the pancreatic beta-cell can be achieved by measuring PI/IRI immediately following acute stimulation of beta-cell secretion, and then determining the proportion of intact proinsulin and proinsulin conversion intermediates contributing to circulating proinsulin-like molecules. In this study, we determined the PI/IRI levels under basal and arginine-stimulated conditions in 17 healthy and 16 NIDDM subjects; high-performance liquid chromatography (HPLC) was also performed in a subset of these subjects to measure the relative contribution of intact proinsulin and its conversion intermediates to total proinsulin-like molecules. In NIDDM subjects, levels of both basal (44.6 +/- 9.6 vs. 9.3 +/- 1.5 pmol/l; P = 0.0007) and stimulated (64.0 +/- 12.7 vs. 19.8 +/- 2.8 pmol/l; P = 0.001) proinsulin-like molecules were higher than in healthy subjects. Although IRI was higher in NIDDM than in control subjects under basal conditions (106 +/- 19 vs. 65.1 +/- 8.1 pmol/l; P = 0.05), it was lower in NIDDM than in control subjects following stimulation (increment: 257 +/- 46 vs. 416 +/- 51 pmol/l; P = 0.03). PI/IRI ratios were increased in NIDDM subjects under both basal (43.3 +/- 5.0 vs. 14.0 +/- 1.3%; P < 0.0001) and stimulated (increment: 10.1 +/- 2.1 vs. 2.5 +/- 0.2%; P = 0.0006) conditions, compatible with the release of a disproportionately increased amount of proinsulin-like products. HPLC analysis revealed that, in the stimulated state, intact proinsulin made up 40.1 +/- 6.7% of proinsulin-like molecules in NIDDM individuals (n = 9) and 30.1 +/- 5.6% in healthy subjects (n = 7; NS). The remainder of the proinsulin-like molecules comprised the des-31,32-split proinsulin conversion intermediate. The increase in PI/IRI in NIDDM under basal and especially under stimulated conditions suggests that proinsulin conversion is indeed perturbed in this disorder. Because the relative proportions of intact and des-31,32-split proinsulin are similar in both healthy and NIDDM subjects, the orderly cleavage of proinsulin at its two junctions appears preserved. However, at the time of exocytosis, the secretory granule in the islet of NIDDM subjects contains an increased proportion of incompletely processed proinsulin, presumably reflecting a slower rate of conversion or granules' reduced time of residence in beta-cells.

Increased susceptibility of islets from diabetes-prone Psammomys obesus to the deleterious effects of chronic glucose exposure.

Patients with noninsulin-dependent diabetes (NIDDM) show an increase in the relative plasma levels of proinsulin and proinsulin conversion intermediates, which is corrected by strict glycemic control. This observation suggests that hyperglycemia per se may be responsible for generating the aberrant plasma hormone profile. The question remains, however, whether a genetic predisposition to NIDDM underlies the failure of the insulin production machinery to meet a prolonged increase in secretory demand. In this study, islet monolayer cultures from the diabetes-prone Psammomys obesus and normal diabetes-resistant rats were exposed to RPMI 1640 medium containing either 11.1 or 33.3 mM glucose; insulin-related peptides were resolved by HPLC. Prolonged exposure (10 days) of rat islets to high glucose resulted in a reduced a secretory response to an acute glucose stimulus associated with a 37% reduction in the insulin content but no change in the proinsulin/insulin ratio. When subjected to a similar protocol, islets from prediabetic Psammomys lost the insulin response to glucose; beta-cell insulin content was reduced by about 70%, and the proportion of proinsulin-related peptides increased from 18% to 38%. In the in vivo situation, pancreatic extracts from nonfasted diabetic Psammomys contained 36% proinsulin-related peptides in contrast to 15% in pancreatic extracts from nondiabetic animals. Thus, prolonged in vitro exposure of prediabetic Psammomys islets to high levels of glucose could reproduce the modified beta-cell secretory profiles observed in vivo in the diabetic animal. These results support the hypothesis that hyperproinsulinemia in NIDDM is secondary to the inability of beta-cells to meet a sustained increase in insulin demand, whereas individuals with normal beta-cells may meet such demand with an adequate output of mature insulin.

Insulin secretion in obese and non-obese NIDDM

Both the insulin response to glucose and the sensitivity to insulin show large variation in the normal population. Many subjects have either a markedly low insulin response or low sensitivity to insulin, with nevertheless normal glucose tolerance. For such subjects to become diabetic, insulin secretion or insulin action must further deteriorate with time, or other factors are added which tip the balance towards diabetes. Most evidence to date indicates that reduced β-cell responsiveness and reduced insulin sensitivity co-exist in subjects prior to developing NIDDM. Both insulin secretion and insulin action are genetically controlled and influenced by intrauterine and neonatal factors. Insulin secretion and insulin action vary inversely in a closely linked manner; inability to fully compensate for changes in one variable mar generate a functional deficit in glucose homeostasis. Subjects combining low functions would run a proportionately larger risk of decompensating the glucose tolerance and be more vulnerable, in terms of diabetes susceptibility, to factors that further reduce insulin output or insulin action. Careful analysis of existing data prompts us to ascribe a dominating role to the impairment of insulin secretion in the pathogenesis of IGT and NIDDM. Patients with NIDDM also exhibit increased proportions of proinsulin and proinsulin conversion intermediates. We used hyperinsulinaemic diabetic and non-diabetic Psammomys obesus to study the possible relationship between steady-state pancreatic insulin stores and the proportion of proinsulin-related peptides in the plasma and the pancreas. A marked increase in these peptides was associated with 90% reduction in insulin stores of the pancreas. After food deprivation, the depletion of pancreatic insulin in the diabetic animals was partially corrected, and the proinsulin z'insulin ratio normalized. In contrast, non-diabetic psammomys showed only 50% reduction in pancreatic insulin stores under non-fasting conditions, with no change in proinsulinyinsulin ratio. These findings suggest that in the diabetic Psammomys obesus, pancreatic capacity for storage/production of insulin is limited; the metabolic consequences of this limitation are amplified by increased secretory demand secondary to insulin resistance, thus facilitating the establishment of hyperglycaemia, which may in itself further exacerbate the pancreatic dysfunction.

Hyperproinsulinemia and insulin deficiency in the diabetic Psammomys obesus

Patients with noninsulin-dependent diabetes mellitus exhibit increased proportions of plasma proinsulin and proinsulin conversion intermediates. We used hyperinsulinemic diabetic and nondiabetic Psammomys obesus to study the possible relationship between steady state pancreatic insulin stores and the proportion of proinsulin-related peptides in the plasma and pancreas. Insulin-like peptides were separated by reverse phase HPLC and identified by pulse-chase experiments. A marked increase in the proportions of proinsulin and proinsulin conversion intermediates in the plasma and pancreas of diabetic nonfasted Psammomys was associated with 90% reduction in insulin stores of the pancreas. After a 16- to 20-h fast, the depletion of pancreatic insulin in the diabetic animals was partially corrected, and the proinsulin/insulin ratio was normalized. In contrast, nondiabetic Psammomys showed only 50% reduction in pancreatic insulin stores under nonfasting conditions, with no change in the proinsulin/insulin ratio. These findings suggest that in the diabetic Psammomys obesus, the pancreatic capacity for storage of insulin may be limited; the metabolic consequences of this limitation are amplified by increased secretory demand secondary to insulin resistance, thus facilitating the establishment of hyperglycemia, which may in itself further exacerbate pancreatic dysfunction.

Hyperproinsulinemia and insulin deficiency in the diabetic Psammomys obesus.

Patients with noninsulin-dependent diabetes mellitus exhibit increased proportions of plasma proinsulin and proinsulin conversion intermediates. We used hyperinsulinemic diabetic and nondiabetic Psammomys obesus to study the possible relationship between steady state pancreatic insulin stores and the proportion of proinsulin-related peptides in the plasma and pancreas. Insulin-like peptides were separated by reverse phase HPLC and identified by pulse-chase experiments. A marked increase in the proportions of proinsulin and proinsulin conversion intermediates in the plasma and pancreas of diabetic nonfasted Psammomys was associated with 90% reduction in insulin stores of the pancreas. After a 16- to 20-h fast, the depletion of pancreatic insulin in the diabetic animals was partially corrected, and the proinsulin/insulin ratio was normalized. In contrast, nondiabetic Psammomys showed only 50% reduction in pancreatic insulin stores under nonfasting conditions, with no change in the proinsulin/insulin ratio. These findings suggest that in the diabetic Psammomys obesus, the pancreatic capacity for storage of insulin may be limited; the metabolic consequences of this limitation are amplified by increased secretory demand secondary to insulin resistance, thus facilitating the establishment of hyperglycemia, which may in itself further exacerbate pancreatic dysfunction.

Highly sensitive enzyme immunoassay of proinsulin immunoreactivity with use of two monoclonal antibodies

A highly sensitive two-site sandwich ELISA measuring total proinsulin immunoreactive material in serum or plasma was developed. The assay was based on two monoclonal antibodies, an anti-C-peptide antibody bound to a microtest plate and a biotin-labeled anti-insulin antibody. The detection limit (3 SD above zero value) in buffer was 0.05 pmol/L, corresponding to 0.25 pmol/L in human serum (diluted 1:5). The linear calibrator range was 0.05-20 pmol/L. Interassay CVs were 4.7% at a median (range) of 2.3 pmol/L (1.4-2.8 pmol/L, n = 8), 6.7% at 5.1 pmol/L (3.3-8.0 pmol/L, n = 8), and 8.7% at 10.0 pmol/L (8-12 pmol/L, n = 10). Mean analytical recovery of added human proinsulin (hPI) (2, 5, and 10 pmol/L) to serum was 84% (range 68-128%, n = 9). Human insulin and human C-peptide did not cross-react at 5000 and 10,000 pmol/L, respectively. The four major proinsulin conversion intermediates reacted 65-99%: split(32-33)hPI 74%, des-(31,32)hPI 65%, split(65-66)hPI 78%, and des(64,65)hPI 99%. All serum values from 38 fasting healthy subjects were above the detection limit: median (range) 4.0 (2.1-12.6) pmol/L.

Proteolytic Degradation of Human Recombinant Proinsulin/Insulin by Sera from Acute Pancreatitis Patients and Complete Inhibition by Eglin-C

Sera from patients of biliary, alcoholic, and idiopathic acute pancreatitis with severity scored from 1 to 5 based on the Ranson criteria were tested for proinsulin/insulin degrading activity. Proinsulin degrading activity by normal controls was 8 +/- 4% as compared with 22-78 +/- 17% with a mean of 45% by the patient sera. An order of magnitude increase of proinsulin degrading activity was accompanied by an order of magnitude increase of immunoreactive pancreatic cationic trypsin(ogen) and (pro)elastase-2 as determined by radioimmunoassay with day 1 sera. Proinsulin degrading activity also showed a negative correlation with the clinical time course and dropped to normal by 6 days after admission. The decrease of proinsulin degrading activity was concomitant with a decrease of serum immunoreactive pancreatic serine proteases. High-performance liquid chromatography analysis of the proteolysis products showed the appearance of insulin and smaller peptides with no proinsulin conversion intermediates. Ninety to ninety-eight percent of proinsulin degrading activity was inhibited by anti-alpha 2-macroglobulin (alpha 2-M) antiserum, or (Ac)Eglin-C(J141), and 52% by an elastase and chymotrypsin-specific inhibitor, MeOSuc-Ala-Ala-Pro-boroVal-pinacol. E64c, TLCK, alpha 1-protease inhibitor (alpha 1-PI), or Trasylol inhibited proinsulin degrading activity by 10-17%, and anti-cathepsin B antiserum by 9%. The observed proinsulin degrading activity did not correlate with the Ranson's scores, age, sex, etiology, total serum immunoreactive insulin, calcium, albumin or alpha 2-M but had a negative correlation with serum alpha 1-PI (r = -0.55) and a positive correlation with serum esterase activity (r = .62).(ABSTRACT TRUNCATED AT 250 WORDS)

Scintigraphic distribution of 123 I labelled proinsulin, split conversion intermediates and insulin in rats.

Insulin, biosynthetic human proinsulin and 2 human proinsulin conversion intermediates, des (64, 65) human proinsulin and des (31, 32) human proinsulin, were labelled with 123 I and the derivatives monosubstituted on Tyr A14 were purified by reverse phase high performance liquid chromatography. The four tracers were injected into anaesthetized rats via a jugular or a portal vein and time activity curves were generated for the liver and kidneys using a gamma camera and an online computer. Liver extraction coefficients varied in the order insulin (38%), des (64, 65) human proinsulin (11.7%), des (31, 32) human proinsulin (3.2%), human proinsulin (1.6%); whereas half-life of hepatic activity varied in the reverse order, from 6 min for insulin, to 45 min for human proinsulin. As expected for a non-receptor mediated process, kidney extraction varied conversely to liver extraction, being highest for human proinsulin and lowest for insulin. It is concluded that the kinetics of human proinsulin conversion intermediates depends upon the site of cleavage and deletion and is intermediate between those of insulin and intact human proinsulin.

A sensitive radioimmunoassay for human proinsulin, with sequential use of antisera to C-peptide and insulin.

In this assay for immunoreactive human proinsulin (IRPI), it first is separated from plasma by use of an antiserum to human C-peptide. An immunoprecipitate is then formed by using a precipitating antiserum and polyethylene glycol, after which IRPI is dissociated from the antiserum by incubation in warm HCl, pH 2.0. The resulting mixture is assayed for insulin immunoreactivity by a double-antibody tracer-competition method involving incubation for four days with a high-affinity anti-insulin antiserum. Human proinsulin of recombinant-DNA origin is used as the standard. Added C-peptide at supraphysiological concentrations did not interfere with or react in the assay. Human insulin cross reacted by 1.5%. The detection limit for IRPI (2 SD from zero-dose binding) is 3 pmol/L. Proinsulin conversion intermediates are measured nearly as well as intact proinsulin. IRPI concentrations in 10 nondiabetic human subjects averaged 12.0 (SEM 1.6) pmol/L. The ratio of proinsulin to immunoreactive insulin averaged 14.3 (SEM 2.2)%. After intravenous arginine, the increase in proinsulin was less than that of insulin, and it declined more slowly.

Biochemical and clinical implications of proinsulin conversion intermediates.

Since a complete map of insulin-related peptides in humans requires consideration of proinsulin, Arg32/Glu33-split proinsulin, Arg65/Gly66-split proinsulin, des-Arg31,Arg32-proinsulin, des-Lys64, Arg65-proinsulin, and insulin, we applied high performance liquid chromatography coupled with radioimmunoassay to investigate the formation of proinsulin conversion intermediates in vitro and in vivo. Kinetic analysis of proinsulin processing by a mixture of trypsin and carboxypeptidase B (to stimulate in vivo processes) revealed (a) a rapid decline in proinsulin concommitant with formation of conversion intermediates, (b) formation of des-Arg31, Arg32-proinsulin and des-Lys64,Arg65-proinsulin in the ratio 3.3:1 at steady state, and (c) complete conversion of the precursor to insulin during extended incubation. Studies on normal human pancreas identified a similar ratio of des-Arg31,Arg32-proinsulin to des-Lys64,Arg65-proinsulin (approximately 3:1), whereas two insulinomas contained sizable amounts of des-Arg31,Arg32-proinsulin, but barely detectable amounts of des-Lys64,Arg65-proinsulin. None of the tissues contained measurable quantities of Arg32/Glu33- or Arg65/Gly66-split proinsulin. Analysis of plasma from three diabetic subjects managed by the intravenous infusion of human proinsulin revealed less than 1% processing of the circulating precursor to conversion intermediates and no processing of the precursor to human insulin. Nevertheless, analysis of plasma from the same subjects managed by the subcutaneous infusion of proinsulin revealed 4-11% processing of the precursor to intermediates that had the properties of des-Arg31,Arg32-proinsulin and Arg65/Gly66-split proinsulin. We conclude that (a) processing of proinsulin to insulin in vivo as in vitro likely occurs by preferential cleavage at the Arg32-Glu33 peptide bond in proinsulin, (b) proinsulin is inefficiently processed in the vascular compartment, and (c) subcutaneous administration of the precursor can result in the formation of conversion intermediates with the potential for contributing to biological activity.

Hyperproinsulinemia in a family with a proposed defect in conversion is linked to the insulin gene.

Two previously described pedigrees with familial hyperproinsulinemia have elevated proinsulin conversion intermediates resulting from amino acid substitutions in the proinsulin molecule. In contrast, a third family with elevated levels of an apparently normal proinsulin molecule may have a defect in the converting process. To determine if the defect in this family lies in the insulin gene region, we used restriction fragment length polymorphisms adjacent to the insulin gene to examine cosegregation with hyperproinsulinemia. We demonstrate linkage of hyperproinsulinemia and the insulin gene in this family with a LOD score of 1.8, suggesting that the defect lies in or near the insulin gene. This method has wide applicability in determining whether hyperproinsulinemia or hyperinsulinemia is the result of defects at the insulin gene, and should permit the detection of new defects at or near this locus.