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PubMed2005

Progressive decrease of proinsulin secretion in sulphonylurea-treated type 2 diabetes.

Progressive deterioration of beta-cell function is proposed as a disease-related factor of sulphonylurea (SU) failure in type 2 diabetes. If it gradually worsens over time then disease duration may mirror the progressive beta-cell deterioration. The aim of the present study is to assess whether or not disease duration is influential in remodelling the secretion pattern of insulin-like molecules and in glucose control of SU-treated type 2 diabetes. A research model is used to investigate proinsulin secreting capacity over time, using two groups of patients: i) disease duration <5 years (n=62), comprising SU responders (SUr; n=48) and SU failures (SUf; n=14); and ii) disease duration > or = 5 years (n= 37), comprising an SUr group (n=17) and an SUf group (n=20). Blood samples are taken at 0 h, 0.5 h 1 h, 2 h and 3 h during a standard oral glucose tolerance test and measured for glucose, total proinsulin (TPI), intact proinsulin (IPI) and specific insulin (SI) concentrations. Pairwise comparison of estimated marginal means of blood glucose, SI, IPI and TPI levels at each time point are carried out between groups and subgroups. (SUr vs. SUf). Homa insulin resistance index (IR index) is applied to analyse IR between the groups. It was found that patients with shorter disease duration had higher proinsulin (TPI and IPI) levels at all time points (P<0.05), together with a lower glucose level at 2 h and 3 h (P<0.05). Homa insulin index analysis showed no difference between the two groups (P=0.26). Results also showed that the SUr group had a significantly lower glucose level at Oh and 3h (P<0.05), although no significant difference in insulin and proinsulin levels was found between the SUr and SUf groups. In conclusion, proinsulin may play an important role in glucose control in SU-treated type 2 diabetes, but the effect is reduced in SUf patients.

Adult↗
PubMed1994

pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle.

By quantitative immunoelectron microscopy and HPLC, we have studied the effect of disrupting pH gradients, by ammonium chloride, on proinsulin conversion in the insulin-producing B-cells of the islets of langerhans. Proinsulin content and pH in single secretory vesicles were measured on consecutive serial sections immunostained alternately with anti-proinsulin or anti-dinitrophenol (to reveal the pH-sensitive probe DAMP) antibodies. Radioactivity labeled proinsulin, proinsulin cleavage intermediates, and insulin were quantitated by HPLC analysis of extracts of islets treated in the same conditions. Cleavage at the C-peptide/A-chain junction is significantly less sensitive to pH gradient disruption than that of the B-chain/C-peptide junction, but the range of pH and proinsulin content in individual vesicles indicate that both cleavages occur in the same vesicle released from the TGN.

Ammonium Chloride↗
PubMed2003

Mutant proinsulin that cannot be converted is secreted efficiently from primary rat beta-cells via the regulated pathway.

Prohormones are directed from the trans-Golgi network to secretory granules of the regulated secretory pathway. It has further been proposed that prohormone conversion by endoproteolysis may be necessary for subsequent retention of peptides in granules and to prevent their release by the so-called "constitutive-like" pathway. To address this directly, mutant human proinsulin (Arg/Gly(32):Lys/Thr(64)), which cannot be cleaved by conversion endoproteases, was expressed in primary rat islet cells by recombinant adenovirus. The handling of the mutant proinsulin was compared with that of wild-type human proinsulin. Infected islet cells were pulse labeled and both basal and stimulated secretion of radiolabeled products followed during a chase. Labeled products were quantified by high-performance liquid chromatography. As expected, the mutant proinsulin was not converted at any time. Basal (constitutive and constitutive-like) secretion was higher for the mutant proinsulin than for wild-type proinsulin/insulin, but amounted to <1% even during a prolonged (6-h) period of basal chase. There was no difference in stimulated (regulated) secretion of mutant and wild-type proinsulin/insulin at any time. Thus, in primary islet cells, unprocessed (mutant) proinsulin is sorted to the regulated pathway and then retained in secretory granules as efficiently as fully processed insulin.

Acetylcysteine↗
PubMed2004

Effects of deletion and shift of the A20-B19 disulfide bond on the structure, activity, and refolding of proinsulin.

To investigate the role of the A20-B19 disulfide bond in the structure, activity and folding of proinsulin, a human proinsulin (HPI) mutant [A20, B19Ala]-HPI was prepared. This mutant, together with another proinsulin mutant previously constructed with an A19Tyr deletion, which can also be taken as shifted mutant of the A20-B19 disulfide bond, were studied for their in vitro refolding, oxidation of free thiol groups, circular dichroism spectra, antibody and receptor binding activities and sensitivity to trypsin digestion in comparison with native proinsulin. The results indicate that deletion of the A20-B19 disulfide bond results in a large decrease in the alpha-helix content of the molecule and higher sensitivity to tryptic digestion. Both the deletion and shift mutations, especially the latter, cause a great decrease in the biological activity of proinsulin analogues. The folding yields of HPI analogues were much lower than that of HPI. And the shift mutant, [Delta A19Tyr]-HPI, was scarcely refolded correctly in vitro and its refolding yield was extremely low. These results suggest that the A20-B19 disulfide bond plays an important role in the structural stabilization and folding of the insulin precursor. By summarizing the refolding studies on proinsulin, a possible folding pathway is proposed.

Binding Sites, Antibody↗
PubMed1994

Epitope analysis of human insulin and intact proinsulin.

Residues belonging to epitopes on human insulin that were recognized by a panel of three monoclonal antibodies were located using mutated insulins and insulins from a number of different animal species. Epitopes on human proinsulin recognized by two monoclonal antibodies were also identified using partially processed proinsulin species. Epitopes were located on the C-A and B-C junctions of proinsulin and on the N-termini of the A- and B-chains and the central region of the B-chain of human insulin. Antibodies that bound proinsulin were found to induce conformational changes in the prohormone. The presence of a well-defined interaction between the C-peptide portion and the N-terminus of the A-chain of the insulin moiety of intact proinsulin has also been demonstrated. The relevance of these studies to the development of two-site assays for the measurement of partially processed proinsulin species in human sera is also discussed.

Animals↗
PubMed1991

Prolonged maximal stimulation of insulin secretion in healthy subjects does not provoke preferential release of proinsulin.

Release of immature secretory granules rich in incompletely processed proinsulin has been proposed to explain the relative hyperproinsulinemia in type 2 diabetic and insulinoma patients because of a constant secretory drive resulting from hyperglycemia and autonomous secretion, respectively. To test this hypothesis, insulin secretion was stimulated by a combination of hyperglycemia (11 mmol/L clamp), intravenous (i.v.) tolbutamide (1 g), and i.v. glucagon (initial bolus 10 micrograms/kg body weight, maintenance infusion 2 micrograms/kg body weight per hour) for 3 h. Circulating IR-insulin and IR-C-peptide concentrations increased 89-fold and 14-fold over basal values, respectively, but IR-proinsulin concentrations increased only ninefold over basal values. Estimation of the amount of insulin secreted (based on deconvolution analysis of plasma C-peptide values) showed that approximately 76 +/- 21 U were secreted during the stimulation period. This amount is a significant proportion of pancreatic insulin content in normal humans. In molar terms, IR-proinsulin (integrated incremental response multiplied by metabolic clearance rate of proinsulin) relative to IR-C-peptide (= insulin) secretion (deconvolution analysis) was estimated to be equal or even lower than the known proportion in islets (0.22 +/- 0.05%). Thus, using a near-maximal stimulation of insulin secretion maintained long enough to cause release of amounts of insulin approaching the estimated pancreatic content, no preferential release of proinsulin was observed in normal humans. Therefore, the hyperproinsulinemia of type 2 diabetes and in insulinoma patients may be caused by additional defects in the proinsulin to insulin conversion process.

Adult↗
PubMed2004

Cross-sectional and prospective association between proinsulin secretion and graft function after clinical islet transplantation.

Proinsulin levels as a marker of beta-cell dysfunction have not been described after clinical islet transplantation. Proinsulin secretion was studied in 23 type 1 diabetic patients after islet allotransplantation and in 20 age-matched nondiabetic controls. Fasting serum insulin, total proinsulin (TP), intact proinsulin, proinsulin fragments (PFs) and their ratios to insulin were determined 1 and 12 months after patients became insulin independent. TP, PF, and proinsulin/insulin ratios were lower in transplant recipients compared with controls, in patients who retained long-term insulin independence. Insulin, C-peptide, and intact proinsulin values were similar in transplant recipients and controls. Hormone levels remained stable over time in the group of patients who retained long-term insulin independence, but the TP and PF levels were higher at 12 months compared with 1 month in the group of patients who resumed insulin therapy. TP and PF levels were reduced in transplant recipients compared with controls but increased over time if insulin independence was lost.

Adult↗
PubMed1988

A direct assay for proinsulin in plasma and its applications in hypoglycaemia.

A direct radioimmunoassay in unextracted plasma is described. The assay has a sensitivity of 4 pmol/l (2 standard deviation from zero). The proinsulin antiserum was immuno-adsorbed against human C-peptide and insulin coupled to glass beads. Cross-reactivity of the antiserum was assessed and shown to be less than 0.01% with both peptides. In normal healthy fasting subjects the plasma proinsulin level was 6.7 +/- 1.7 pmol/l (n = 17) (mean +/- SD). Fasting proinsulin levels in non-insulin dependent diabetics were significantly elevated compared with non diabetics (14.2 +/- 2 pmol/l (n = 11) vs 6.7 +/- 1.7 (n = 17) P less than 0.005). The insulin/proinsulin ratio was 3.4:1 in the non-insulin dependent diabetic compared with 6:1 in non-diabetics. Samples from 21 insulinoma patients were assayed and mean fasting plasma proinsulin level was 255 pmol/l +/- 479 when the patients were hypoglycaemic. The range in pro-insulin levels was large (30-2300 pmol/l). Mean fasting proinsulin level in three hypoglycaemic subjects due to sulphonylurea overdose was 15.7 +/- 2.3 pmol/l. The molar ratio of proinsulin to insulin was 1:6 in healthy subjects, 1:1 in insulinoma patients and 10:1 in sulphonylurea induced hypoglycaemic patients.

C-Peptide↗
PubMedDate not supplied

Insulin therapy in type 2 diabetic subjects suppresses plasminogen activator inhibitor (PAI-1) activity and proinsulin-like molecules independently of glycaemic control.

Eleven Type 2 diabetic subjects (10 male 1 female: age 56.2 +/- 9.7 (SD) yr) were treated in random order either with insulin or with sulphonylureas for 8 weeks each, without attempting to alter glycaemic control between the two treatment periods. Insulin treatment was associated with suppression of endogenous insulin secretion (fasting C-peptide levels -35.0 +/- 24.2%; p = 0.006), and of intact proinsulin (-43.1 +/- 36.8%; p = 0.03) and 32,33 split proinsulin -20.1 +/- 27.0%; p = 0.03). Activity of plasminogen activator inhibitor (PAI-1), a fast acting inhibitor of fibrinolysis, decreased significantly (-14.3% +/- 27.5%; p = 0.02) but no changes occurred in concentration of lipoproteins or apoproteins between therapies. Changes in concentrations of 32,33 split and intact proinsulin were closely and significantly related (rs = 0.83; p < 0.001) to each other but not with changes in concentrations of C-peptide (intact proinsulin rs = -0.41; p = 0.11) and 32,33 split proinsulin rs = -0.27; (p = 0.21). Percentage changes in intact proinsulin concentrations were positively correlated with those in PAI-1 (rs = 0.51; p = 0.05). There was, however a paradoxical negative relationship between changes in C-peptide concentrations and those of PAI-1 (rs = -0.73; p = 0.006). These preliminary observations suggest that insulin treatment in Type 2 diabetic subjects without any changes in glycaemic control is associated with a reduced activity of PAI-1, but is without effect on any other cardiovascular risk factors. Concentrations of insulin precursor molecules may play a role in determining fibrinolytic activity.

Apolipoproteins↗
PubMed1994

Fetal proinsulin and birth weight.

In both diabetic and non-diabetic pregnancies fetal insulin is an important anabolic hormone. Fetal hyperinsulinaemia is associated with accelerated fetal growth and increased birth weight. Insulin and C-peptide concentrations in both umbilical cord and amniotic fluid reflect fetal beta-cell secretion and are correlated with birth weight. In the present study umbilical venous proinsulin and insulin concentrations were measured in 54 term infants born to women with and without mild disturbances of glucose tolerance. Umbilical venous cord proinsulin, assayed using a highly specific immunoradiometric assay, was independently correlated with infant birth weight (Rho = 0.53, p < 0.0001) and birth percentile (Rho = 0.65, p < 0.0001). The correlation between birth weight and birth percentile weight with umbilical venous insulin, measured using a non-specific insulin assay, was lost following correction for the influence of proinsulin. Umbilical venous cord proinsulin appears to be a good indicator of fetal beta-cell activity, and in this study, a superior marker to insulin assayed using a non-specific insulin radioimmunoassay. The longer half-life of proinsulin compared with insulin may contribute to proinsulin being a more robust marker of overall fetal beta-cell activity than insulin.

Adult↗
PubMed1977

Cell-free modulation of proinsulin synthesis.

In vivo, glucose preferentially stimulates proinsulin biosynthesis; at least part of this process is independent of new RNA synthesis and is accompanied by increases in the overall rate of polypeptide chain initiation. The cell-free translation of proinsulin messenger RNA is very sensitive to changes in the protein-synthesizing system. Proinsulin synthesis is preferentially inhibited by the addition of increasing quantities of polyadenylate-containing RNA from the fetal bovine pancreas or by the addition of the drug, aurintricarboxylic acid, which blocks polypeptide chain initiation. These results suggest that proinsulin messenger RNA completes less efficiently for rate controlling initiation factors. We propose that glucose stimulates proinsulin biosynthesis by allowing the less competitive proinsulin messenger RNA to be translated more efficiently.

Aurintricarboxylic Acid↗
PubMed1989

Proinsulin mRNA levels in fasting and fed ADX rats: evidence for an indirect effect of glucocorticoids.

To determine whether glucocorticoids are required to maintain pancreatic proinsulin mRNA levels during dietary manipulation, rats were adrenalectomized (ADX) or sham operated (SO) and subsequently fasted or pair fed for 2 days. Proinsulin mRNA concentrations were 54 +/- 8% lower (P less than 0.05) in fed ADX rats and 47 +/- 10% lower (P less than 0.01) in fasted ADX rats relative to values in fed and fasted SO rats, respectively. When ADX rats were fasted for 24 h and either refed 20% sucrose for 30 h or injected with dexamethasone (DEX) 0.125 mg/kg ip every 12 h for three doses, circulating plasma glucose levels were restored and pancreatic proinsulin mRNA concentrations rose 3.3 and 2.7-fold, respectively (each P less than 0.05). Plasma glucose and proinsulin mRNA levels (n = 40) were correlated (r = 0.58, P less than 0.0001). We conclude that the regulation of proinsulin mRNA concentration does not absolutely require endogenous glucocorticoids, since either adequate sucrose intake in ADX rats or physiological glucocorticoid responses in fasted rats suffice to restore pancreatic mRNA concentrations. It appears that glucocorticoid stimulation of pancreatic proinsulin mRNA levels is mediated indirectly through its regulation of glucose metabolism.

Adrenalectomy↗
PubMed1995

Insulin, intact and split proinsulin, and coronary artery disease in young men.

BACKGROUND: Glucose intolerance and hyperinsulinemia are common disturbances in nondiabetic men with premature coronary artery disease (CAD). To investigate the relation between insulin-like molecules and severity of coronary atherosclerosis, 62 consecutive nondiabetic men presenting with a first myocardial infarction before the age of 45 were studied along with 41 healthy, age-matched, male, population-based control subjects. METHODS AND RESULTS: Specific two-site immunoradiometric assays were used to distinguish intact proinsulin, (des 31,32) proinsulin, and "true" insulin in fasting plasma and during an oral glucose tolerance test (OGTT). Global coronary atherosclerosis and number and severity of distinct stenoses were determined in the patients in 15 proximal coronary arterial segments by use of separate semiquantitative classification systems. The patients had a two- to threefold increase in insulin and insulin propeptide concentrations in the fasting state as well as during the OGTT. Severity of coronary atherosclerosis correlated significantly (P < .05 to P < .01) with basal proinsulin (r = .40) and the proinsulin area under the curve (AUC) (r = .34), basal insulin (r = .31), basal C peptide (r = .30), and the glucose AUC (r = .30). In multiple stepwise regression analysis including insulin-like molecules, major plasma lipoproteins, and lipoprotein subfractions, basal proinsulin (increase in R2 = .09) and dense LDL triglycerides (increase in R2 = .10) predicted 19% of the variation of the global coronary atherosclerosis score after adjustment for age, body mass index, fasting insulin concentration, and VLDL triglycerides. CONCLUSIONS: This study shows that young, nondiabetic, male survivors of myocardial infarction are truly hyperinsulinemic during an OGTT and suggests a close association between proinsulin and coronary atherosclerosis.

Adrenergic beta-Antagonists↗
PubMed2002

Sex differences in the association between proinsulin and intact insulin with coronary heart disease in nondiabetic older adults: the Rancho Bernardo Study.

BACKGROUND: Insulin or insulin resistance is considered a coronary heart disease (CHD) risk factor, but proinsulin may have a stronger association with CHD than insulin. The role of sex differences in this association is unclear. We examined the cross-sectional association of proinsulin and insulin with CHD in older men and women without diabetes. METHODS AND RESULTS: A cross-sectional study of community-dwelling men (n=554) and women (n=902), 50 to 97 years of age, without diabetes by history or oral glucose tolerance test, was done between 1992 and 1996; plasma levels of intact insulin, proinsulin, and C-peptide were measured by radioimmunoassay. Based on questionnaire, medical history, or ECG abnormalities, 25% of men (n=136) and 24% of women (n=214) had prevalent CHD. All insulin variables were positively correlated with CHD risk factors. Compared with those without CHD, men and women with CHD had significantly higher levels of proinsulin. Women but not men with CHD also had higher levels of C-peptide and fasting and postchallenge insulin. Only proinsulin was significantly and independently associated with prevalent CHD in both men (OR=2.41, 1.42 to 4.11) and women (OR=1.80, 1.22 to 2.64) (adjusted for age, body mass index, systolic blood pressure, and HDL cholesterol). Similar analyses for fasting and postchallenge intact insulin and for C-peptide showed that among these three variables, only postchallenge insulin was significantly associated with CHD, and only in women. CONCLUSIONS: In older nondiabetic men and women, proinsulin was more strongly and consistently associated with CHD than was intact insulin.

Aged↗
PubMed1975

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.

Antigens↗
PubMed1987

A soluble-phase proinsulin radioimmunoassay and its use in diagnosis of hypoglycaemia.

A soluble-phase proinsulin assay has been developed which does not require solid-phase antibody-binding. A human proinsulin standard curve is prepared in insulin-free and proinsulin-free plasma for comparison with unknown plasma samples. Proinsulin and insulin are bound with excess anti-insulin antiserum, and free C-peptide is removed by charcoal adsorption. The supernatant is then assayed using a routine C-peptide radioimmunoassay which utilises anti-C-peptide antiserum. The sensitivity of the assay (2 standard deviations above zero) is 9 pmol/L using 200 microL plasma sample. The assay is free from insulin cross-reactivity up to 100 mU/L and C-peptide up to 2000 pmol/L. Between-assay CV is 13% at 100 pmol/L. The assay has been used in subjects with hypoglycaemia of various aetiologies and has shown that a raised plasma proinsulin in the presence of hypoglycaemia can occur in sulphonylurea-induced and reactive hypoglycaemia as well as in insulinomas. After hyperglycaemic clamps at 7.5, 10 and 15 mmol/L glucose, type II diabetics both on and off sulphonylurea, were found to have lower proinsulin concentrations compared with normal subjects, commensurate with the diabetics' lower insulin responses.

Adult↗
PubMed1995

Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells.

Endoproteolytic cleavage of pairs of basic amino acids is the key mechanism in the specific processing of precursor hormone molecules. Two endoproteases, PC1 (or PC3) and PC2, have recently been implicated in the conversion of proinsulin. Using antibodies against these proteases and proinsulin, followed by protein A-gold complex, we performed an immunocytochemical study for precise identification of the subcellular compartments involved in the processing of insulin. Both PC1 and PC2 immunoreactivities followed a pattern of gradually increasing density along the secretory pathway, being higher in the immature granules. Proinsulin labeling was detected in the Golgi apparatus and in the coated immature secretory granules located mainly in the Golgi area. Using double labeling, we demonstrated the presence of PC1 and/or PC2 in the majority of proinsulin-rich granules. In addition, we provided evidence that PC1 and PC2 are co-localized within the same granules. Co-expression of PC1 and PC2 with proinsulin in islet beta-cells indicates that these proteases are actively involved, probably in a sequential manner, in the conversion of proinsulin into insulin.

Animals↗
PubMed1995

Possibilities of false immunocytochemical results generated by the use of monoclonal antibodies: the example of the anti-proinsulin antibody.

The immunocytochemical application of a monoclonal antibody (MAb) against the Arg-Arg region at the junction of the C-peptide and the insulin beta-chain of the human proinsulin molecule on rat pancreatic tissue resulted in positive immunogold labeling over the insulin- as well as the glucagon-secreting cells. In the insulin cells, the Golgi cisternae and the immature clathrin-coated granules were intensely labeled, and in the glucagon cells all the granules were labeled. Control experiments using the anti-proinsulin MAb adsorbed with proinsulin, insulin, and glucagon have confirmed the crossreactivity of this proinsulin antibody with glucagon. Furthermore, the anti-proinsulin MAb appears to crossreact not only with rat but also with human glucagon and with bovine and porcine insulin and glucagon. Examination of the amino acid sequences of proinsulin and proglucagon has shown that both molecules display this Arg-Arg dipeptide sequence, which could explain the labeling obtained on both cell types. In addition, other propeptides also display such a sequence and, indeed, positive cells were detected in rat pituitary. These results demonstrate that by immunocytochemistry this MAb, although providing very specific results, reveals non-related molecules. This is due to the fact that these molecules exhibit similar short determinants. MAbs raised against very short amino acid sequences can reveal this determinant in various molecules, generating false-positive results.

Animals↗