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PubMed1992

Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implication of a favored route for prohormone processing.

Two Ca(2+)-dependent endopeptidase activities are involved in proinsulin to insulin conversion: type I cleaves COOH-terminal to proinsulin Arg31-Arg32 (B-chain/C-peptide junction); and type II preferentially cleaves at the Lys64-Arg65 site (C-peptide/A-chain junction). To further understand the mechanism of proinsulin processing, we have investigated types I and II endopeptidase processing of intact proinsulin in parallel to that of the conversion intermediates, des-31,32-proinsulin and des-64,65-proinsulin. The type I processed des-64,65-proinsulin and proinsulin at the same rate. In contrast, the type II endopeptidase processed des-31,32-proinsulin at a much faster rate (> 19-fold; p < 0.001) than it did intact proinsulin. Furthermore, unlabeled proinsulin concentrations required for competitive inhibition of 125I-labeled des-64,65-proinsulin and 125I-proinsulin processing by a purified insulin secretory granule lysate were similar (ID50 = 14-16 microM), whereas inhibition of 125I-labeled des-31,32-proinsulin processing required a higher nonradiolabeled proinsulin concentration (ID50 = 197 microM). Synthetic peptides corresponding to the sequences surrounding Lys64-Arg65 (AC-peptide/substrate) and Arg31-Arg32 (BC-peptide/substrate) of human proinsulin were synthesized for use as specific substrates or competitive inhibitors. Cleavage of the BC-substrate by type I and AC-substrate by type II was COOH-terminal of the dibasic sequence, with similar Ca(2+)-and pH requirements previously observed for proinsulin cleavage. Apparent Km and Vmax for type I processing of the BC-substrate was Km = 20 microM; Vmax = 22.8 pmol/min, and for type II processing of the AC-substrate was Km = 68 microM; Vmax = 97 pmol/min. In competitive inhibition assays, the BC-peptide similarly blocked insulin secretory granule lysate processing of des-64,65-proinsulin and proinsulin (ID50 = 45-55 microM), but did not inhibit des-31,32-proinsulin processing. However, the AC-peptide preferentially inhibited insulin secretory granule lysate processing of des-31,32-proinsulin (ID50 = microM) compared to proinsulin (ID50 = 330 microM), and not des-64,65-proinsulin. We conclude that the type I endopeptidase recognized des-64,65-proinsulin and proinsulin as similar substrates, whereas the type II endopeptidase has a stronger preference for des-31,32-proinsulin compared to intact proinsulin. Furthermore, we suggest that in intact proinsulin there exists a constraint to efficient processing that is relieved following type I processing. Structural flexibility, in addition to the presence of Lys64-Arg65, therefore appears to be important for type II endopeptidase specificity and may provide a molecular basis for a preferential route of proinsulin conversion via des-31,32-proinsulin.

Amino Acid Sequence↗
PubMed1989

Sensitive and specific two-site immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split proinsulins.

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.

Adult↗
PubMed1997

The relation of proinsulin, insulin, and proinsulin-to-insulin ratio to insulin sensitivity and acute insulin response in normoglycemic subjects.

Plasma levels of proinsulin and its conversion intermediates are elevated in NIDDM patients. Recent studies have suggested that proinsulin levels are also increased relative to insulin levels in subjects who subsequently develop NIDDM. This may be due to insulin resistance or a defect in proinsulin processing or insulin secretion. If insulin resistance is the trigger, the proinsulin-to-insulin ratio would be higher in insulin-resistant subjects than in insulin-sensitive subjects. We examined the association of fasting proinsulin, 32,33 split proinsulin, and the proinsulin-to-insulin ratio with insulin sensitivity (SI), estimated by a frequently sampled intravenous glucose tolerance test and the minimal model in 138 normoglycemic subjects ages 53-61 years. We also investigated the relation of proinsulins and the proinsulin-to-insulin ratio to acute insulin response (AIR). Fasting specific insulin (r = -0.64), intact proinsulin (r = -0.43), and 32,33 split proinsulin (r = -0.54) concentrations were inversely correlated and the proinsulin-to-insulin ratio positively (r = 0.31) correlated with SI (P < 0.001). Fasting specific insulin (r = 0.64), intact proinsulin (r = 0.35), and 32,33 split proinsulin (r = 0.45) concentrations were positively correlated and proinsulin-to-insulin ratio (r = -0.40) inversely correlated with AIR (P < 0.001). The proinsulin-to-insulin ratio increased by increasing levels of SI (quartiles of SI from low to high: 0.048, 0.078, 0.078, 0.068; P = 0.012) and decreased by increasing AIR (quartiles of AIR from low to high: 0.088, 0.068, 0.058, 0.058; P = 0.005). These associations were independent of age, sex, BMI, and waist-to-hip ratio. Furthermore, the relation between the proinsulin-to-insulin ratio and AIR was independent of SI. In conclusion, in normoglycemic subjects, insulin resistance (low SI) was associated with a low rather than a high proinsulin-to-insulin ratio. Subjects who maintained normoglycemia with a low AIR had an increased proinsulin-to-insulin ratio compared with those who needed high AIR to maintain normoglycemia. These results suggest that, in subjects with normal glucose tolerance, insulin resistance does not induce increased proinsulin relative to insulin secretion, but rather is associated with enhanced processing of proinsulin.

Blood Glucose↗
PubMed1989

Immunoradiometric assay of human proinsulin and partially processed proinsulin with use of monoclonal antibody and streptavidin-biotin labeling.

The sensitive and specific immunoradiometric assay is described for human proinsulin and its intermediate peptides (65-66 split and 32-33 split proinsulin). We developed a monoclonal antibody-based two-site immunoradiometric assay with use of streptavidin-biotin labeling. The detection limits of the assays lie in the range of 0.5-2.0 pM. In the proinsulin assay proinsulin cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. In the assay of 65-66 split proinsulin it does not cross-react with insulin, proinsulin or 32-33 split proinsulin. In the 32-33 split proinsulin assay it cross-reacted 84% with proinsulin and 60% with 65-66 split proinsulin. The precision (C.V.) of the assays was less than 15% over the various concentration. The mean concentrations of insulin, proinsulin, 65-66 split proinsulin and 32-33 proinsulin in eight young male subjects in the fasting state were (pM +/- S.E.M.) 20 +/- 3.6, 2.3 +/- 0.3, undetectable (less than 1.0) and 2.1 +/- 0.7 and at the maximum reached during an oral glucose tolerance test, 150 +/- 26, 9.9 +/- 1.4, 3.8 +/- 0.6 and 19.7 +/- 6.0 respectively.

Adult↗
PubMed1993

The effects of subcutaneous human proinsulin on the production of 64/65 split proinsulin, glucose turnover and intermediary metabolism in non-insulin-dependent diabetic man.

We have compared the effects of subcutaneously injected human proinsulin, insulin zinc suspension and inactive diluent (control) on glucose turnover, intermediary carbohydrate and lipid metabolism in non-insulin-dependent diabetic man. Six weight-matched (24.8 +/- 1.6 kg M-2) non-insulin-dependent diabetic subjects underwent 3 separate, randomized, 10 h isoglycemic clamps. Glucose turnover was measured using a primed continuous infusion of [6'6'2H2] glucose. Each subject received 0.35 U/kg of hormone or control made up to isovolumetric amounts. The mean blood glucose level of 7.3 +/- 0.8 mmol/l was similar at the start of each isoglycemic clamp. Incremental area under the curve proinsulin levels (1195 +/- 146 nmol/l) were about 21-fold higher, on a molar basis, than insulin (62.4 +/- 10 nmol/l). Des 64/65 split proinsulin increased in a parallel manner to intact proinsulin (r = 0.99, P < 0.0001) and comprised approximately 13% of the intact proinsulin concentration. Hepatic glucose production was suppressed similarly following proinsulin and insulin zinc injection. However, both proinsulin and insulin zinc had a significantly greater effect on suppression of hepatic glucose production compared to control (P = 0.01, P = 0.009, respectively). Metabolic clearance rate of glucose fell significantly during the control studies compared to insulin zinc or proinsulin injections (P < 0.05). Blood lactate, pyruvate and alanine concentrations were similar following control or hormone injections. However blood glycerol, 3-hydroxybutyrate and plasma-non-esterified fatty acids were suppressed significantly by proinsulin and insulin zinc compared to control injections. The conclusions were: (1) In overnight fasted hyperglycemic non-insulin-dependent subjects s.c. injections of proinsulin and insulin zinc can produce similar effects on glucose turnover, intermediary lipid and carbohydrate metabolism. (2) Similar carbohydrate intermediary metabolism profiles can be obtained following insulin zinc, proinsulin or control injections. (3) However lipolysis and ketogenesis were significantly suppressed by both hormones compared to control. (4) Subcutaneous proinsulin injection resulted in approximately 13% conversion to des 64/65 split proinsulin.

Alanine↗
PubMed1996

Glucose-regulated translational control of proinsulin biosynthesis with that of the proinsulin endopeptidases PC2 and PC3 in the insulin-producing MIN6 cell line.

In the short term (< 2 h), proinsulin biosynthesis is predominately glucose regulated at the translational level; however, the details at the molecular level behind this mechanism are not well defined. One of the major hindrances for gaining a better understanding of the proinsulin biosynthetic mechanism has been a lack of an abundant source of beta-cells that express a phenotype of regulated proinsulin biosynthesis in the appropriate 2.8-16.7 mmol/l glucose range as defined in normal pancreatic islets. In this study, we demonstrate that in the MIN6 cell line, specific glucose-regulated translational control of proinsulin biosynthesis is present in the appropriate glucose concentration range. In addition to that of proinsulin, the biosynthesis of the two proinsulin conversion endopeptidases, PC2 and PC3, was coordinately glucose regulated in MIN6 cells, whereas that of the exopeptidase, carboxypeptidase H, was unaffected by glucose. Proinsulin, PC2 and PC3 biosynthesis was specifically stimulated over that of total MIN6 cell protein synthesis above a threshold of 4 mmol/l glucose that reached a maximum rate between 8 and 10 mmol/l glucose. Glucose-induced proinsulin, PC2, and PC3 biosynthesis was rapid (occurring after a 20-min lag period but reaching a maximum by 60 min), unaffected by the presence of actinomycin D; and in parallel experiments, stimulatory glucose concentrations did not alter MIN6 cell total preproinsulin, PC2, or PC3 mRNA levels. Thus, short-term (< 2 h) glucose stimulation of proinsulin, PC2 and PC3 biosynthesis in MIN6 cells, like that in isolated islets, was mediated at the translational level. Intracellular signals for mediating glucose-stimulated proinsulin PC2 and PC3 biosynthesis translation in MIN6 cells also appeared to be similar to those in pancreatic islets, requiring glucose metabolism and a supporting role for protein kinase A. However, protein kinase C or a Ca(2+)-dependent protein kinase did not appear to be required for glucose-regulated proinsulin biosynthesis in MIN6 cells, as in islets. MIN6 cells are the first beta-cell line that indicate glucose-regulated proinsulin biosynthesis translation essentially identical to that in differentiated islet beta-cells and will be an important experimental model to better define the mechanism of proinsulin biosynthesis in detail.

Animals↗
PubMed2003

Insulin, proinsulin, proinsulin:insulin ratio, and the risk of developing type 2 diabetes mellitus in women.

PURPOSE: To assess the associations among baseline levels of fasting insulin and proinsulin, proinsulin:insulin ratio, and the development of type 2 diabetes mellitus in apparently healthy middle-aged women. METHODS: In a nested case-control study involving a nationwide cohort of 27,628 participants from the Women's Health Study, 126 women with diabetes diagnosed during a 4-year follow-up period were compared with 225 age-matched controls. Fasting insulin level and proinsulin:insulin ratio were assessed in quartiles, and proinsulin level was assessed in categories (< or =4.0 pmol/L, 4.01 to 6.99 pmol/L, > or =7.0 pmol/L). The risk of developing type 2 diabetes was determined using conditional logistic regression analysis that adjusted for body mass index and other diabetes risk factors. RESULTS: Baseline insulin and proinsulin levels and proinsulin:insulin ratios were significantly higher among cases than among controls. Women with elevated insulin levels in the highest as compared with the lowest quartile were more likely to develop diabetes (odds ratio [OR] = 5.6; 95% confidence interval [CI]: 1.8 to 17.6), as were women with elevated (> or =7.0 pmol/L vs. < or =4.0 pmol/L) proinsulin levels (OR = 16.4; 95% CI: 5.8 to 46.8) and women with proinsulin:insulin ratios in the highest quartile (OR = 9.6; 95% CI: 3.1 to 30.8). Similar results were observed among women with a baseline hemoglobin A(1c) level < or =6.0%. In time-trend analyses, fasting insulin was a consistent predictor of long-term risk. Proinsulin and proinsulin:insulin ratio, although predictive throughout the study, were especially strong predictors of rapid progression to type 2 diabetes. CONCLUSION: Elevated fasting insulin and proinsulin levels and proinsulin:insulin ratio are associated with an increased risk of developing type 2 diabetes in apparently healthy middle-aged women.

Biomarkers↗
PubMed1999

Effects of antihyperglycaemic therapies on proinsulin and relation between proinsulin and cardiovascular risk factors in type 2 diabetes.

AIM: To assess the effect of oral antihyperglycaemic therapy on fasting proinsulin and the relation between proinsulin levels and cardiovascular risk factors in type 2 diabetes. METHODS: One hundred and sixty-five patients with type 2 diabetes, fasting blood glucose concentration (FBG) > or = 6.7 mmol/l, were recruited from five diabetes outpatient clinics in primary health care. Diet and antihyperglycaemic medication, aiming at FBG < 6.7 mmol/l, was maintained for 6 months after completed dose titration in a randomized, double-blind, double-dummy trial with metformin (M), glibenclamide (G) and primary combination of both drugs (MG). The study compared M, G and MG in low dose (MGL) and also different high-dose regimens, i.e. G added to M (M/G), M added to G (G/M) and primary combination (MGH). Outcome measures were fasting proinsulin, glycaemia, body mass index, blood pressure, lipids, insulin and C-peptide. RESULTS: Lower proinsulin levels were found when therapy was initiated with metformin (M vs. G, p = 0.013 and M/G vs. G/M, p = 0.033). M and G were equally effective on glucose levels. In the group as a whole FBG decreased from (mean +/- s.d.) 10.2 +/- 2.7 to 7.0 +/- 1.2 mmol/l with no change in proinsulin. Proinsulin was associated with cardiovascular risk factors, linking high proinsulin to an atherogenic risk marker profile. Mean proinsulin change from baseline was inconsistently associated with markers of insulin resistance. Meal-stimulated glucose (net AUC) decreased after treatment only in those with low baseline proinsulin levels. CONCLUSION: It may be advantageous to initiate oral antihyperglycaemic therapy with metformin rather than with sulphonylurea. High proinsulin levels are associated with an atherogenic-risk marker profile and an impaired therapeutic postprandial glucose response after treatment in patients with type 2 diabetes. Proinsulin change after therapy is inconsistently associated with markers of insulin resistance and unrelated to fasting blood glucose reduction.

Biomarkers↗
PubMed2002

Severe block in processing of proinsulin to insulin accompanied by elevation of des-64,65 proinsulin intermediates in islets of mice lacking prohormone convertase 1/3.

The neuroendocrine processing endoproteases PC2 and PC1/3 are expressed in the beta cells of the islets of Langerhans and participate in the processing of proinsulin to insulin and C-peptide. We have previously shown that disruption of PC2 (SPC2) expression significantly impairs proinsulin processing. Here we report that disruption of the expression of PC1/3 (SPC3) produces a much more severe block in proinsulin conversion. In nulls, pancreatic and circulating proinsulin-like components comprise 87% and 91%, respectively, of total insulin-related immunoreactivity. Heterozygotes also show a more than 2-fold elevation in proinsulin levels to approximately 12%. Immunocytochemical and ultrastructural studies of the beta cells reveal the nearly complete absence of mature insulin immunoreactivity and its replacement by that of proinsulin in abundant immature-appearing secretory granules. In contrast, alpha cell morphology and glucagon processing are normal, and there is also no defect in somatostatin-14 generation. Pulse-chase labeling studies confirm the existence of a major block in proinsulin processing in PC1/3 nulls with prolongation of half-times of conversion by 7- and 10-fold for proinsulins I and II, respectively. Lack of PC1/3 also results in increased levels of des-64,65 proinsulin intermediates generated by PC2, in contrast to PC2 nulls, in which des- 31,32 proinsulin intermediates predominate. These results confirm that PC1/3 plays a major role in processing proinsulin, but that its coordinated action with PC2 is necessary for the most efficient and complete processing of this prohormone.

Animals↗
PubMed1998

Incomplete processing of proinsulin to insulin accompanied by elevation of Des-31,32 proinsulin intermediates in islets of mice lacking active PC2.

The prohormone convertases PC2 (SPC2) and PC3/PC1 (SPC3) are the major precursor processing endoproteases in a wide variety of neural and endocrine tissues. Both enzymes are normally expressed in the islet beta cells and participate in proinsulin processing. Recently we generated mice lacking active PC2 due to a disruption of the PC2 gene (Furuta, M., Yano, H., Zhou, A., Rouillé, Y., Holst, J. J., Carroll, R. J., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646-6651). Here we report that these PC2 mutant mice have elevated circulating proinsulin, comprising 60% of immunoreactive insulin-like components. Acid ethanol extractable proinsulin from pancreas is also significantly elevated, representing about 35% of total immunoreactive insulin-like components. These increased amounts of proinsulin are mainly stored in secretory granules, giving rise to an altered appearance on electron microscopy. In pulse-chase experiments, the mutant islets incorporate lesser amounts of isotopic amino acids into insulin-related components than normal islets. In both wild-type and mutant islets, proinsulin I was processed more rapidly to insulin, reflecting the preference of both PC2 and PC3 for substrates having a basic amino acid positioned four residues upstream of the cleavage site. The overall half-time for the conversion of proinsulin to insulin is increased approximately 3-fold in the mutant islets and is associated with a 4-5-fold greater elevation of des-31,32 proinsulin, an intermediate that is formed by the preferential cleavage of proinsulin at the B chain-C-peptide junction by PC3 and is C-terminally processed to remove Arg31 and Arg32 by carboxypeptidase E. The constitutive release of newly synthesized proinsulin from both mutant and wild-type islets during the first 1-2 h of chase was normal (<2% of total). These results demonstrate that PC2 plays an essential role in proinsulin processing in vivo, but is quantitatively less important in this regard than PC3, and that its absence does not influence the efficient sorting of proinsulin into the regulated secretory pathway.

Amino Acid Sequence↗
PubMed1993

Proinsulin, proinsulin intermediate and insulin in cystic fibrosis.

OBJECTIVE: We determined the changes in insulin, intact proinsulin and 32-33 split proinsulin as markers of beta cell damage in assessing the state of carbohydrate intolerance in patients with cystic fibrosis. DESIGN: Measurements of insulin, proinsulin and 32-33 proinsulin concentrations in cystic fibrosis following oral glucose tolerance test (OGTT). SUBJECTS: Sixteen cystic fibrosis patients attending the chest outpatient clinic for follow-up, age range 14-42 years, and 14 healthy controls matched for age and body mass index. MEASUREMENTS: Insulin, intact proinsulin and 32-33 split proinsulin were measured immunoradiometrically at 0, 30, 60, 90 and 120 minutes after an OGTT. Glucose and HbA1 were also measured. RESULTS: Cystic fibrosis patients were divided into two groups according to their OGTT results: those with diabetes mellitus or impaired glucose tolerance and those with normal glucose tolerance. Insulin concentrations and insulin/glucose ratios at 30 minutes were lower in both cystic fibrosis groups in comparison with the control. There was also a significant increase in the time to reach peak insulin levels in both cystic fibrosis groups. Fasting intact proinsulin concentrations and the proportion of proinsulin-like molecules were significantly higher in cystic fibrosis with diabetes mellitus or impaired glucose tolerance than in the control group, but not in the normal glucose tolerance cystic fibrosis group. There was no significant difference in the plasma concentrations of 32-33 split proinsulin amongst the three groups. CONCLUSIONS: Abnormal beta cell function in cystic fibrosis patients was reflected initially in a diminished 30-minute insulin response to oral glucose. A significant rise in fasting intact proinsulin and the proportion of proinsulin-like molecules was seen only in cystic fibrosis patients who had progressed to impaired glucose tolerance or diabetes mellitus. Cystic fibrosis patients with normal glucose tolerance showed changes intermediate between the control and the other group.

Adolescent↗
PubMed1985

Measurement of circulating human proinsulin concentrations using a proinsulin-specific antiserum.

Antibodies have been raised against biosynthetic human proinsulin that show less than 1% cross-reactivity with human insulin and C-peptide. A sensitive (IC50 0.16 pmol/ml; minimum detectable concentration 0.004 pmol/ml) radioimmunoassay has been developed using this antiserum and 125I-proinsulin that will measure proinsulin-like immunoreactivity in human serum without the need for prior separation of insulin or C-peptide. In healthy, fasted subjects (N = 23), the serum proinsulin concentration was 0.015 +/- 0.001 pmol/ml (mean +/- SEM). In six healthy subjects, serum proinsulin rose to 250% of basal after 120 min in response to 100 g oral carbohydrate, but to only 130% after 60 min following 25 g oral carbohydrate. The proinsulin/total immunoreactive insulin ratio and the proinsulin/C-peptide ratio fell sharply after both high and low carbohydrate loads. Endogenous human serum proinsulin-like immunoreactivity released into the circulation after 100 g carbohydrate was eluted from a Mono Q high-performance, ion-exchange column with the same retention time as biosynthetic human proinsulin. Treatment of biosynthetic proinsulin with trypsin under mild conditions led to a decrease in proinsulin-like immunoreactivity concomitant with an increase in C-peptide and insulin-like immunoreactivity, indicating that the proinsulin-specific antiserum did not preferentially recognize intermediates of proinsulin cleavage.

Adult↗
PubMed1986

The effects of proinsulin pretreatment on the combined actions of insulin and proinsulin in normal man.

With the recent availability of biosynthetic human proinsulin there has been a renewed interest in evaluating its metabolic effects, either alone or in combination with insulin. It has been suggested that pretreatment with proinsulin enhances the hypoglycemic response to subsequently administered insulin. On the other hand, the simultaneous administration of proinsulin and insulin has additive, not synergistic, effects. To clarify this question we used the euglycemic glucose clamp technique in 10 normal subjects to compare the steady state effects on glucose disposal of combined infusions of insulin (0.54 microgram/M2 . min, equivalent to 15 mU/M2 . min) and proinsulin (2.75 micrograms/M2 . min) given both simultaneously and sequentially. The mean +/- SEM steady state glucose disposal rates were similar whether the two hormones were given simultaneously (7.2 +/- 0.7 mg/min . kg), after proinsulin pretreatment (7.7 +/- 0.7 mg/min . kg), or after insulin pretreatment (7.1 +/- 0.7 mg/min . kg). The serum proinsulin concentration of 5.39 +/- 0.3 pmol/ml during the infusion of proinsulin alone was unchanged by the simultaneous infusion of insulin, suggesting that in the doses used, insulin did not affect proinsulin clearance. We conclude that in normal subjects there is no enhancement of the combined action of insulin and proinsulin to stimulate glucose disposal by pretreatment with proinsulin or insulin.

Adult↗
PubMed1988

Binding of proinsulin and proinsulin conversion intermediates to human placental insulin-like growth factor I receptors.

Insulin-like growth factor I (IGF-I) and proinsulin share similarities in both primary and tertiary structure. Proinsulin, endogenously secreted or exogenously administered, would, therefore, be expected to interact with IGF-I receptors. We determined the relative activities of IGF-I, insulin, proinsulin, and the proinsulin conversion intermediates in IGF-I radioreceptor assays using term human placental membranes. Insulin was approximately 0.5% as potent as IGF-I, and proinsulin was only 2% as potent as insulin. The six major proinsulin conversion intermediates were studied; all had activities intermediate between those of insulin and proinsulin. We conclude that the binding of proinsulin and the proinsulin conversion intermediates to IGF-I receptors is not of physiological significance at the concentrations occurring endogenously or after exogenous administration of proinsulin.

Binding, Competitive↗
PubMedDate not supplied

Abnormal proinsulin levels in thyroid dysfunction measured by a sensitive proinsulin immunochemiluminoassay.

Abnormalities of carbohydrate metabolism in hyperthyroid patients have been long noted. Elevated proinsulin level is considered as an early marker of B-cell impairment. Proinsulin levels in hyperthyroid patients decreased after antithyroid drug therapy. However, proinsulin in hypothyroid patients was only rarely reported, and the difference was only demonstrated after glucose stimulation-there was a greater response of proinsulin secretion after thyroxine therapy-and the basal fasting proinsulin level was not different after therapy. One of the reasons might be that the assay was not sensitive enough to detect the change of basal proinsulin levels in patients with hypothyroidism after therapy. A newly developed immunochemiluminometric assay of proinsulin was used to demonstrate that the suppressed proinsulin level increased after thyroxine therapy in hypothyroid patients (4.2 +/- 2.4 vs. 10.0 +/- 5.6 pmol/L, p < 0.05; n = 7). On the other hand, our study also confirmed that the proinsulin levels decreased in hyperthyroid patients after antithyroid therapy by methimazole (27.8 +/- 26.0 vs. 15.8 +/- 15.7 pmol/L, p < 0.05; n = 12). In conclusion, proinsulin increased in hypothyroid patients after thyroxine therapy and decreased in hyperthyroid patients after methimazole therapy. The results demonstrated there is a high correlation between thyroid function and B-cell function in hypothyroid as well as hyperthyroid patients.

Adult↗
PubMed1996

Effect of metformin on intact proinsulin and des 31,32 proinsulin concentrations in subjects with non-insulin-dependent (type 2) diabetes mellitus.

We have investigated the effects of metformin treatment on concentrations of proinsulin-like molecules in subjects with Type 2 (non-insulin-dependent) diabetes mellitus. Metformin was given for 12 weeks in an increasing dose up to 850 mg three times daily in a double-blind placebo-controlled cross-over design to 27 subjects (age 53.0 +/- 9.9 years; 19 male, 8 female). Concentrations of insulin and proinsulin-like molecules were measured by highly specific enzymoimmunometric assays. The end of metformin treatment was compared with end of placebo treatment. Metformin lowered fasting plasma glucose concentrations (at 12 weeks, metformin: 8.0 +/- 2.5 vs placebo: 12.0 +/- 2.3 mmol l-1, p r2 0.001;). Concentrations of intact (median change -2.9 (range -28.4 to +2.5 pmol l-1), p = 0.02) and des 31,32 proinsulin (median change -1.6 (range -14.1 to +5.4 pmol l-1), p = 0.07) and percentage of proinsulin-like molecules were reduced by metformin treatment (median change -6% (range -16% to +6%), p = 0.02). Changes in the ratio of proinsulin-like molecules were significantly related with those in fasting plasma glucose (r1 = 0.69, p < 0.001). Changes in concentrations of intact and des 31,32 proinsulin on metformin were not related to changes in body mass index or fasting glucose concentration or changes in concentrations of total triglyceride, cholesterol, and plasminogen activator inhibitor-1. Therefore, metformin treatment in subjects with Type 2 diabetes mellitus significantly reduced concentrations of proinsulin-like molecules over a 12-week period. However, these changes were not related to changes in cardiovascular risk factors seen during metformin treatment. We conclude that short-term effects of metformin treatment on proinsulin-like molecules are similar to those previously observed with dietary treatment in subjects with Type 2 diabetes but opposite to those of sulphonylurea treatment. The effect of long-term treatment with metformin on proinsulin-like molecules needs to be assessed.

Blood Glucose↗
PubMed1991

Differential rates of conversion of rat proinsulins I and II. Evidence for slow cleavage at the B-chain/C-peptide junction of proinsulin II.

Rat proinsulin I is converted into insulin more rapidly than is proinsulin II. To study this further, rat islets were labelled (10 min) and conversion kinetics of the labelled proinsulins were monitored during a 120 min chase. Proinsulins, conversion intermediates and both insulins were separated by h.p.l.c. The accumulation of des-64,65-(split proinsulin II) during the chase suggests that the B-chain/C-peptide junction of proinsulin II is cleaved more slowly than the equivalent site on proinsulin I. This accounts for the differential kinetics of conversion of proinsulins I and II, and is presumed to be caused by one (or more) of the amino acid replacements which distinguish the two proinsulins.

Animals↗
PubMed1996

Hyperproinsulinemia in a three-generation Caucasian family due to mutant proinsulin (Arg65-His) not associated with imparied glucose tolerance: the contribution of mutant proinsulin to insulin bioactivity.

Familial hyperproinsulinemia is a genetic abnormality characterized by an increased proportion of proinsulin immunoreactivity in the circulation due to mutations affecting the posttranslational processing of proinsulin. In affected Japanese families, this has been associated with noninsulin-dependent diabetes mellitus or impaired glucose tolerance. A three-generation Caucasian family with hyperproinsulinemia was identified through unexplained hyperinsulinemia in a normal volunteer participating in a metabolic study. High pressure liquid chromatography analysis of fasting plasma revealed a major peak eluting close to the position of proinsulin. Direct sequencing of the proinsulin gene exon 3 showed a heterozygous point mutation (CGT-->CAT) resulting in the substitution of Arg-->His in position 65 (corresponding to the AC cleavage site) in the index case, his mother, and his maternal grandmother. Using specific enzyme-linked immunosorbent assay methods to quantify insulin and proinsulin (including its conversion intermediates), the impact of this mutation on B cell secretion and glucose tolerance was studied. All affected subjects had normal oral glucose tolerance. In the basal state and after oral glucose administration, their proinsulin responses were immense, but intact insulin responses were slightly reduced. However, when calculating insulin bioactivity by assuming 9% activity for mutant Arg65-->His proinsulin, responses in affected subjects were comparable to those in normal subjects. In conclusion, our data demonstrate hyperproinsulinemia in a three-generation Caucasian family due to heterozygous mutant Arg65-->His proinsulin. This was not associated with impaired glucose tolerance. These results suggest that this mutation in the heterozygous state per se does not affect glucose tolerance and that the biological activity of mutant proinsulin contributes to glucose homeostasis in this family. The association of the same mutation with impaired glucose tolerance or diabetes in previous studies may be the result of selection bias or associated conditions (e.g. the genetic background of the kindreds examined).

Adult↗