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PubMed1994

Effect of modification of connecting peptide of proinsulin on its export.

The production of a high yield of proinsulin using a secretion vector has been difficult, even with such modifications to the vector as a strong promoter and a good ribosome binding site. This investigation was therefore initiated to see whether modification of the connecting peptide of proinsulin has any effect on the export of proinsulin. We constructed three types of proinsulin secretion vectors: (a) pEZZ18-PI, by inserting the proinsulin gene into pEZZ18 vector; (b) pEZZ18-PI-C, by modifying ZZ-proinsulin by addition of the carboxy terminal peptide region of human insulin-like growth factor I (hIGFI) to the carboxy terminal end of proinsulin; and (c) pEZZ18-PI analogues, by sequentially deleting the connecting peptide region of proinsulin. The highest export yield of proinsulin was obtained when the connecting peptide region of the proinsulin was similar in size to that of hIGFI, or when most of the connecting peptide region of the proinsulin was deleted. The amount of exported ZZ-proinsulin analogues in these clones was over 25-times higher than that of ZZ-proinsulin with an unmodified connecting peptide in the secretion/expression vector pEZZ18-PI. On the basis of these observations, we conclude that modification of the mature domain of proinsulin is a critical factor for determination of the export of proinsulin.

Amino Acid Sequence↗
PubMed1970

The biological and immunological properties of pork and beef insulin, proinsulin, and connecting peptides.

The recently discovered hormone precursors, pork and beef proinsulins, their respective connecting peptides, and beef proinsulin intermediates have been compared to insulin in their ability to stimulate the conversion of glucose-U-(14)C to (14)CO(2) and lipids in isolated fat cells. The concentrations of beef and pork proinsulins required to achieve the same biological effect were respectively, 15 and 10 times that of insulin. Beef proinsulin intermediates required only 2.6 times the concentration of insulin for the same effect. Pork and beef connecting peptides in high or low concentrations alone or in combination with proinsulin, insulin, or proinsulin intermediates showed no biological effect on the isolated fat cell system. The insulin-like activity of beef and pork proinsulins on the isolated fat cell system was not abolished with pancreatic trypsin or kallikrein inhibitors. Pork insulin antiserum inhibited the biological activity of pork insulin and proinsulin as well as that of beef insulin or proinsulin. Pork proinsulin antiserum also inhibited the insulin-like activity of both pork insulin and proinsulin. By the radioimmunoassay method, pork insulin antiserum bound only (1/4) to [unk] as much proinsulin as insulin. Beef proinsulin intermediates, on the other hand, were found to react with the pork insulin antiserum to an extent nearly equal to that of insulin. These data suggest that (a) proinsulin exhibits its effect on the isolated fat cells independent of its conversion to insulin, (b) connecting peptides have no biological effect under present experimental conditions, and (c) in comparison to insulin, immunological reactivity of proinsulin is greater than its biological activity using our pork insulin antiserum; thus, the comparison of antibody specificity with the fat cell receptor specificity suggests that the biological site of action is different from the immunologic site.

Adipose Tissue↗
PubMed1998

Proinsulin and insulin concentrations in relation to carotid wall thickness: Insulin Resistance Atherosclerosis Study.

BACKGROUND AND PURPOSE: Insulin resistance and hyperinsulinemia have been associated with atherosclerosis. Recent attention has focused on the possible role of proinsulin because most radioimmunoassays for insulin cross-react with proinsulin. Therefore, it is not known which of the two, insulin per se or proinsulin, is more strongly related to atherosclerosis. METHODS: We examined the relation between fasting proinsulin, fasting split proinsulin, fasting and 2-hour insulin (after oral glucose load), and intima-media wall thickness (IMT) in the common carotid artery (CCA) and internal carotid artery (ICA) in 985 nondiabetic subjects from the Insulin Resistance Atherosclerosis Study, a multiethnic study of insulin resistance and atherosclerosis. RESULTS: In the overall population, a weak but significant relation between proinsulin and CCA IMT was observed (r=0.07, P=0.029). However, the relation between proinsulin and IMT was stronger in Hispanics and non-Hispanic whites than in African Americans. In non-Hispanic whites and Hispanics, significant correlations between CCA and proinsulin (r=0.087) and between ICA and proinsulin (r=0.101), split proinsulin (r = 0.092), and fasting insulin (r = 0.087) were observed. The significant correlations became more attenuated (and nonsignificant) after adjustment for cardiovascular risk factors, especially plasminogen activator inhibitor-1 (PAI-1). CONCLUSIONS: The association between proinsulin and IMT, while weak, appears to be stronger than the association between insulin and IMT. Adjustment for PAI-1 markedly attenuated the association between proinsulin and IMT, suggesting a possible mediating role for PAI-1 in this association. It is possible that proinsulin may represent a marker of atherosclerosis rather than a causal factor for atherosclerosis. Studies of the insulin resistance syndrome and atherosclerosis that use insulin as a surrogate for insulin resistance should consider the use of specific insulin assays as well as determination of proinsulin concentrations.

Arteriosclerosis↗
PubMed2003

Acceleration of type 1 diabetes mellitus in proinsulin 2-deficient NOD mice.

Accumulating evidence favors a role for proinsulin as a key autoantigen in diabetes. In the mouse, two proinsulin isoforms coexist. Most studies point to proinsulin 2 as the major isoform recognized by T cells in the NOD mouse. We studied mice in which a null proinsulin 2 mutation was transferred from proinsulin 2-deficient 129 mice onto the NOD background along with 16 genetic markers (including I-A(g7) MHC molecule) associated with diabetes. Intercross mice from the fourth backcross generation showed that proinsulin 2(-/-) mice develop accelerated insulitis and diabetes. The high prevalence of anti-insulin autoantibodies in proinsulin 2(-/-) mice indicates that diabetes acceleration relates to altered recognition of proinsulin. The prevalence of anti-glutamic acid decarboxylase autoantibodies and of sialitis is not increased in proinsulin 2(-/-) mice. We give evidence that proinsulin 2 expression leads to silencing of T cells specific for an epitope shared by proinsulin 1 and proinsulin 2. In the human, alleles located in the VNTR region flanking the insulin gene control beta cell response to glucose and proinsulin expression in the thymus and are key determinants of diabetes susceptibility. Proinsulin 2(-/-) NOD mice provide a model to study the role of thymic expression of insulin in susceptibility to diabetes.

Adoptive Transfer↗
PubMed2003

Clinical and laboratory evaluation of specific chemiluminescence assays for intact and total proinsulin.

Measurement of proinsulin is an important tool in the assessment of pancreatic beta cell function in patients with type 2 diabetes. The goal of this study was to perform a technical and clinical evaluation of two specific chemiluminescence assays (CLIA) for the determination of intact and total proinsulin in comparison to a radioimmunoassay (RIA) method for the measurement of total proinsulin. A total of 191 serum samples from patients with type 2 diabetes were used to perform a regression analysis. The total proinsulin CLIA showed higher proinsulin levels than the two other proinsulin assays (mean +/- SD: 55.9 +/- 58.1 pmol/l, p < 0.001 in both cases). The intact proinsulin CLIA (22.5 +/- 20.9 pmol/l) gave lower values than the RIA for total proinsulin (31.9 +/- 25.4 pmol/l, p < 0.001 vs. CLIA, r = 0.948). The RIA has a 95% cross-reactivity to des31,32-proinsulin, which is secreted during the process of beta cell deterioration. The intact proinsulin CLIA has virtually no cross-reactivity with des31,32-proinsulin (1.4%) and is therefore more specific for intact proinsulin than the RIA. This test does not measure further degradation products, in contrast to the total proinsulin CLIA. The CLIA is, therefore, more specific for total proinsulin measurement than the RIA. Both CLIAs could be performed much faster (4 hours) than the RIA method (75 hours/ 4 days). In conclusion, the CLIA methods show improved qualitative outcomes, higher specificity and several technical advantages over the RIA method.

Clinical Laboratory Techniques↗
PubMed1984

In vitro characterization of biosynthetic human proinsulin.

The binding and biologic properties of human proinsulin produced by recombinant DNA technology have been determined. The biosynthetic human proinsulin was iodinated using lactoperoxidase and subsequently purified by HPLC to yield the [(125I)TyrA14]-proinsulin isomer. Using isolated rat adipocytes, biosynthetic human proinsulin was shown to have approximately 11% of the binding potency of native insulin. At 16 degrees C and 37 degrees C, the ED50 values of biosynthetic human proinsulin were 3.7 nM and 15 nM, respectively, which was significantly different from the insulin values of 0.4 nM and 1.7 nM, respectively. Kinetic analysis suggested that the decreased affinity of biosynthetic human proinsulin was due primarily to a decreased association rate rather than an increased dissociation rate. Similar to insulin, biosynthetic human proinsulin exhibited a decreased half-time of dissociation in the presence of insulin (16.7 nM) or proinsulin (111 nM); however, this negative cooperative effect was lost in the presence of high concentrations of proinsulin (11 microM). Biologic potency, assessed by measuring glucose transport in rat adipocytes, showed that biosynthetic human proinsulin had 10% of the biologic activity of insulin, suggesting close coupling between binding to receptors and membrane generated cellular response. By extracting cell surface bound proinsulin with acidic buffer, the amount of 125I-proinsulin that internalized following binding to surface receptors was measured. At equilibrium, 55% of the cell-associated radioactivity was internalized at 37 degrees C. When chloroquine-treated (200 microM) cells were incubated with 125I-proinsulin at 37 degrees C, a 1.5-fold increase in the amount of intracellular proinsulin was observed at 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Processing of proinsulin by furin, PC2, and PC3 in (co) transfected COS (monkey kidney) cells.

The enzymology of proinsulin conversion was studied in COS cells by cotransfection of three species of proinsulin and each of three conversion endoproteases (furin, PC2, and PC3). In addition to the parts of basic residues linking the B-chain to C-peptide (Arg31-Arg32) and C-peptide to the A-chain (Lys64-Arg65), which were present in all three proinsulins studied, human proinsulin presents a P4 basic residue (four residues NH2-terminal to the point of cleavage) only at the former junction (Lys29) and rat proinsulin II only at the latter (Arg62). Human proinsulin Arg62 (prepared by site-directed mutagenesis of human proinsulin) contains a P4 basic residue at both junctions. Transfected cells were incubated for four successive 2-h periods. The media were pooled, and pro-insulin, conversion intermediates, and insulin were separated by reverse-phase high-performance liquid chromatography to monitor conversion activity. There was little conversion of any proinsulin in COS cells without cotransfection of an exogenous endoprotease. When furin or PC3 was cotransfected with any of the three proinsulins, there was extensive processing, with insulin as the major conversion product. PC2, by contrast, failed to cleave human proinsulin but was able to cleave both human proinsulin Arg62 and rat proinsulin II. Cleavage by PC2 of these proinsulins was predominantly at the C-peptide-A-chain junction, generating the conversion intermediate des-64,65-split proinsulin as the major product and only very small amounts of insulin itself.

Amino Acid Sequence↗
PubMed1988

Determination of proinsulin intermediates by means of an immunoradiometric method using polyclonal antibodies.

In our endeavour to develop a method for proinsulin determination, an immunoradiometric method was developed. Thereby guinea-pig antibodies to bovine insulin which were purified with an immunoadsorbent, were, in surplus, coupled to polyethylene tubes. These tubes were used to extract proinsulin and insulin from the sample and standard that or was to be determined. The proinsulin adsorbed onto the wall of the tube was distinguished from insulin by incubating, in a second step, a rabbit antibody to human C-peptide with the tubes. In order to render the proinsulin anti-C-peptide complex measurable, a donkey antibody to rabbit IgG was used, which had been purified via an immunoadsorbent and which was labelled with iodine-125. Since proinsulin extracted from human pancreata was available next to biosynthetic human proinsulin, it was striking to note that these substances were very differently recorded by the determination method applied. Thus biosynthetic human proinsulin dissolved in gelatin buffer could not be measured at all. After mild tryptic cleavage of the biosynthetic human proinsulin, a clear increase of the immunoreactivity was seen in this method. Therefore the claim could be made that partially cleaved proinsulin molecules with a retained C-peptide structure had come into existence. This could be verified by application of the proinsulin cleavage products 65/A1 and 32/33, which exhibited a behaviour very similar to that of pancreatic proinsulin in this method. In this way we could demonstrate that the originally planned immunoradiometric determination method for proinsulin, recorded partially hydrolized intermediates of the proinsulin, which represent a large part of the proinsulin immunoreactivity in the serum.

Antibodies↗
PubMed1999

The relation of proinsulin and insulin to insulin sensitivity and acute insulin response in subjects with newly diagnosed type II diabetes: the Insulin Resistance Atherosclerosis Study.

AIMS/HYPOTHESIS: Proinsulin concentrations are increased relative to insulin concentrations in subjects with Type II (non-insulin-dependent) diabetes mellitus. This could be secondary to hyperglycaemia or insulin resistance or due to a defect in insulin secretion. METHODS: We investigated the association between fasting insulin, intact proinsulin and the intact proinsulin: insulin ratio with insulin sensitivity, estimated by a frequently sampled intravenous glucose tolerance test and the minimal model and with acute insulin response (AIR) in 182 newly diagnosed Type II diabetic subjects aged 40 to 69 years. None of the subjects was receiving hypoglycaemic medication. RESULTS: Insulin sensitivity correlated inversely with fasting insulin (r(s) = -0.42) and intact proinsulin (r(s) = -0.32) (p < 0.001). The intact proinsulin:insulin ratio was not correlated with insulin sensitivity. AIR correlated positively with intact proinsulin (r(s) = 0.23) and inversely with the intact proinsulin:insulin ratio (r(s) = -0.29, p < 0.001). Fasting glucose correlated positively with intact proinsulin (r(s) = 0.34) and the intact proinsulin:insulin ratio (r(s) = 0.24, p < 0. 001). The intact proinsulin:insulin ratio increased by decreasing AIR (quartiles of AIR from high to low: 7.8, 8.2, 9.7 and 12.1 %, p < 0.001). This association was independent of age, sex, ethnicity, body mass index, fasting glucose, and insulin sensitivity. CONCLUSION/INTERPRETATION: Insulin resistance (low insulin sensitivity) was not related to the intact proinsulin:insulin ratio in subjects with Type II diabetes. In contrast, both low AIR and high fasting glucose concentrations were associated with a disproportionate increase in proinsulin concentration. These results suggest that increased intact proinsulin:insulin ratio is a marker of a defect in insulin secretion in Type II diabetic subjects.

Adult↗
PubMed1998

A distinct difference in the metabolic stimulus-response coupling pathways for regulating proinsulin biosynthesis and insulin secretion that lies at the level of a requirement for fatty acyl moieties.

The regulation of proinsulin biosynthesis in pancreatic beta-cells is vital for maintaining optimal insulin stores for glucose-induced insulin release. The majority of nutrient fuels that induce insulin release also stimulate proinsulin biosynthesis, but since insulin exocytosis and proinsulin synthesis involve different cellular mechanisms, a point of divergence in the respective metabolic stimulus-response coupling pathways must exist. A parallel examination of the metabolic regulation of proinsulin biosynthesis and insulin secretion was undertaken in the same beta-cells. In MIN6 cells, glucose-induced proinsulin biosynthesis and insulin release shared a requirement for glycolysis to generate stimulus-coupling signals. Pyruvate stimulated both proinsulin synthesis (threshold 0.13-0.2 mM) and insulin release (threshold 0.2-0.3 mM) in MIN6 cells, which was eliminated by an inhibitor of pyruvate transport (1 mM alpha-cyano-4-hydroxycinnamate). A combination of alpha-oxoisohexanoate and glutamine also stimulated proinsulin biosynthesis and insulin release in MIN6 cells, which, together with the effect of pyruvate, indicated that anaplerosis was necessary for instigating secondary metabolic stimulus-coupling signals in the beta-cell. A consequence of increased anaplerosis in beta-cells is a marked increase in malonyl-CoA, which in turn inhibits beta-oxidation and elevates cytosolic fatty acyl-CoA levels. In the beta-cell, long-chain fatty acyl moieties have been strongly implicated as metabolic stimulus-coupling signals for regulating insulin exocytosis. Indeed, it was found in MIN6 cells and isolated rat pancreatic islets that exogenous oleate, palmitate and 2-bromopalmitate all markedly potentiated glucose-induced insulin release. However, in the very same beta-cells, these fatty acids in contrast inhibited glucose-induced proinsulin biosynthesis. This implies that neither fatty acyl moieties nor beta-oxidation are required for the metabolic stimulus-response coupling pathway specific for proinsulin biosynthesis, and represent an early point of divergence of the two signalling pathways for metabolic regulation of proinsulin biosynthesis and insulin release. Therefore alternative metabolic stimulus-coupling factors for the specific control of proinsulin biosynthesis at the translational level were considered. One possibility examined was an increase in glycerophosphate shuttle activity and change in cytosolic redox state of the beta-cell, as reflected by changes in the ratio of alpha-glycerophosphate to dihydroxyacetone phosphate. Although 16.7 mM glucose produced a significant rise in the alpha-glycerophosphate/dihydroxyacetone phosphate ratio, 1 mM pyruvate did not. It follows that the cytosolic redox state and fatty acyl moieties are not necessarily involved as secondary metabolic stimulus-coupling factors for regulation of proinsulin biosynthesis. However, the results indicate that glycolysis and the subsequent increase in anaplerosis are indeed necessary for this signalling pathway, and therefore an extramitochondrial product of beta-cell pyruvate metabolism (that is upstream of the increased cytosolic fatty acyl-CoA) acts as a key intracellular secondary signal for specific control of proinsulin biosynthesis by glucose at the level of translation.

Animals↗
PubMed1997

Proinsulin and its conversion intermediates in human pancreas and isolated islet tissue: kinetics and steady-state analysis.

In non-insulin-dependent diabetes, circulating insulin-related immunoreactivity (IRI) is often composed of a higher fraction of the incompletely converted forms proinsulin and des-31,32 proinsulin. The present study describes an immunoadsorption method for measuring the proportions of proinsulin, its two split products, and insulin in human pancreatic tissue and for determining their rates of formation in human isolated islets. The method uses two junction-specific monoclonal proinsulin antibodies in a protein G fractionation; it is validated by > or = 90% specificity and recovery. The peptide contents measured in tissue extracts were comparable to those determined in a previously developed immunoradiometric assay. In the nine tissue extracts from nondiabetic donor organs, 97% of IRI corresponded to insulin, 1% to proinsulin, 2% to the des-31,32 proinsulin conversion product, and 0.1% to des-64,65 proinsulin. Two samples from non-insulin-dependent diabetics under sulfonylurea treatment contained a fourfold lower content of IRI but the peptide distribution was comparable except for a low percentage (0.3) of proinsulin in one case. In pulse-chase experiments on three-preparations of human islets isolated from nondiabetic donors, proinsulin represented the major (> 90%) IRI that was synthesized at the end of the 30-min pulse; a subsequent 90-min chase at either 2.5 or 10 mM glucose resulted in conversion of 75% of proinsulin to des-31,32 (20%) and des-64,65 (2%) intermediates and to insulin (50%); after a 180-min chase, 88% of proinsulin was converted to insulin, but 10% remained present as proinsulin. In a pulse-chase experiment on islets isolated from tissue with a high proportion of des-31,32 intermediate (5% instead of 2%), the conversion process was slower (45% after 90 min and 70% after 180 min) and resulted in a higher fraction of des-31,32 intermediate, suggesting that the elevated tissue content in this intermediate is caused by a reduced PC2 converting activity. These data confirm that des-31,32 proinsulin represents the major conversion intermediate in normal human islets and indicate the existence of slow converters, possibly as a result of decreased enzymatic processing of the prohormone's AC junction.

Adolescent↗
PubMed2004

Abnormalities of proinsulin processing in functioning insulinomas: clinical implications.

OBJECTIVE: Abnormal proinsulin processing in insulinomas may result in secretory granules containing both insulin and proinsulin, a finding not encountered in healthy beta-cells. The aim of this study was to test whether such abnormalities in the proinsulin to insulin conversion have clinical implications in patients with hypoglycaemic disorders. DESIGN: Case-series. PATIENTS AND METHODS: Fifteen patients with histologically confirmed insulinoma and two patients with islet cell hyperplasia were included. The immunohistochemical distribution pattern of proinsulin within the tumour cells was classified as Golgi pattern (predominantly perinuclear immunolabelling) or diffuse pattern (immunolabelling in the periphery of the cells, indicating the presence of proinsulin in secretory granules). Data obtained from the 72-h fast and arterial calcium stimulation and hepatic venous sampling (ASVS) test were related to the morphological classification. RESULTS: Six insulinomas exhibited a diffuse proinsulin distribution pattern, while nine insulinomas and two islet cell hyperplasias disclosed a Golgi pattern. Median proinsulin concentrations at the termination of the fast tended to be higher in patients with the diffuse proinsulin distribution pattern than in patients with the Golgi pattern (86.9 vs. 18.8 pmol/l, P = 0.07). Higher insulin (P < 0.005) and proinsulin (P < 0.05) concentrations were significantly correlated with earlier occurrence of hypoglycaemia during the prolonged fast. During the ASVS test, tumours with the diffuse proinsulin distribution pattern exhibited a higher increase in both insulin (median, 37.3- vs. 10.5-fold, P < 0.05) and proinsulin (6.3- vs. 1.6 fold, P < 0.005) concentrations following calcium stimulation than the tumours with the Golgi pattern. CONCLUSIONS: Abnormalities in the proinsulin to insulin conversion in patients with insulinomas and islet cell hyperplasia correlate with impaired regulation of both insulin and proinsulin secretion during the prolonged fast as well as the ASVS test.

Adult↗
PubMed1995

Measurement of proinsulin and intermediates. Validation of immunoassay methods by high-performance liquid chromatography.

Human proinsulin and 32-33 split proinsulin have been measured in the peripheral circulation by immunoradiometric assays (IRMAs) and have been shown to be elevated in impaired glucose tolerance and non-insulin-dependent diabetes mellitus (NIDDM). The IRMA for 32-33 split proinsulin did not discriminate between this molecule and des-32 or des-31,32 split proinsulin. We describe the comparison of IRMA for human plasma proinsulin and 32-33 split proinsulins with assays combined with high-performance liquid chromatography (HPLC), which can discriminate between 32-33 split, des-32 split, and des-31,32 split proinsulin. Subjects were those with normal glucose tolerance (n = 8) and those with NIDDM (n = 17), who were studied while fasting and 30 min after a glucose load. After collection, blood was centrifuged promptly, and the serum/plasma was stored frozen until assay. Both IRMA and HPLC methods were calibrated against synthetic peptides. Interassay coefficients of variation for the IRMA for proinsulin and 32-33 split proinsulin were < 13% over the ranges 3.8-65 pmol/l and 6.4-65 pmol/l, respectively. The following regression lines were obtained: proinsulin IRMA = -0.143 + 1.066 HPLC, r = 0.860; 32-33 split proinsulin IRMA = 0.048 + 1.051 HPLC; and des-31,32 split proinsulin, r = 0.814. For both analytes, there was no significant difference in the relationship of IRMA to HPLC results between the various subject groups and various time points. Thus, the IRMA for proinsulin has been validated by an independent method.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2003

Insulin does not mediate glucose stimulation of proinsulin biosynthesis.

It has recently been suggested that insulin augments its own production by a physiologically important feed-forward autocrine loop. We studied the kinetics of glucose-regulated proinsulin gene expression and proinsulin biosynthesis in normal rat islets with emphasis on the potential role of insulin as a mediator of the glucose effect. There was a time-dependent increase in steady-state proinsulin mRNA in islets cultured at 16.7 mmol/l compared with 3.3 mmol/l glucose; no early (1-3 h) increase in proinsulin gene expression was observed. In contrast, there was a threefold increase in proinsulin biosynthesis within 1 h of glucose stimulation that was not affected by inhibition of glucose-stimulated proinsulin gene transcription with actinomycin D. In addition, inhibition of glucose-stimulated insulin secretion with diazoxide had no effect on glucose-stimulated proinsulin mRNA or biosynthesis. Furthermore, addition of different concentrations of insulin to islets cultured in low glucose failed to affect proinsulin biosynthesis. Taken together, our data suggest that the early glucose-dependent increase in proinsulin biosynthesis is mainly regulated at the translational level, rather than by changes in proinsulin gene expression. Moreover, we could not demonstrate any effect of insulin on islet proinsulin mRNA level or rate of proinsulin biosynthesis. Thus, if insulin has any effect on the proinsulin biosynthetic apparatus, it is a minor one. We conclude that the secreted insulin is not an important mediator of insulin production in response to glucose.

Animals↗
PubMed2006

Tolerance to proinsulin-2 is due to radioresistant thymic cells.

Proinsulin is a key Ag in type 1 diabetes, but the mechanisms regulating proinsulin immune tolerance are unknown. We have shown that preproinsulin-2 gene-deficient mice (proins-2(-/-)) are intolerant to proinsulin-2. In this study, we analyzed the mechanisms underlying T cell-mediated tolerance to proinsulin-2 in 129/Sv nonautoimmune mice. The expression of one proinsulin-2 allele, whatever its parental origin, was sufficient to maintain tolerance. The site of proinsulin-2 expression relevant to tolerance was evaluated in thymus and bone marrow chimeras. CD4+ T cell reactivity to proinsulin-2 was independent of proinsulin-2 expression in radiation-sensitive bone marrow-derived cells. A wt thymus restored tolerance in proins-2(-/-) mice. Conversely, the absence of the preproinsulin-2 gene in radioresistant thymic cells was sufficient to break tolerance. Although chimeric animals had proinsulin-2-reactive CD4+ T cells in their peripheral repertoire, they displayed no insulitis or insulin Abs, suggesting additional protective mechanisms. In a model involving transfer to immunodeficient (CD3epsilon(-/-)) mice, naive and proinsulin-2-primed CD4+ T cells were not activated, but could be activated by immunization regardless of whether the recipient mice expressed proinsulin-2. Furthermore, we could not identify a role for putative specific T cells regulating proinsulin-2-reactive CD4+ T in transfer experiments. Thus, proinsulin-2 gene expression by radioresistant thymic epithelial cells is involved in the induction of self-tolerance, and additional factors are required to induce islet abnormalities.

Adoptive Transfer↗
PubMed1988

Pharmacokinetics of biosynthetic human proinsulin following intravenous and subcutaneous administration in metabolically healthy volunteers.

40 pmol of biosynthetic human proinsulin was administered to 8 healthy volunteers by intravenous and by subcutaneous route. Following proinsulin administration, venous blood was collected in regular intervals within which proinsulin was determined by a specific radioimmunometric assay with monoclonal antibodies. The proinsulin concentration was determined simultaneously with the insulin and C-peptide radioimmunoassay. Through this investigation the following kinetic parameters were found: The kinetics of the biosynthetic human proinsulin can be best described by the 3-compartment model. The dominant biological half-life was 92 minutes. In intravenous proinsulin administration a proinsulin mean transit time of 80 minutes was found, whereas in subcutaneous administration a proinsulin retention time of 225 minutes was measured. The mean resorption velocity of the subcutaneously applied proinsulin amounted to 145 minutes. Two lag times for subcutaneous resorption can be described, a short one with 9.4 minutes and a long one with 65 minutes. The initial distribution volume for proinsulin was 3.8 l, whereas the distribution volume after complete distribution was 9.3 l. The mean total metabolic clearance was determined with 120 ml/min. Since no difference for the proinsulin concentration was found using the 3 different determination methods a peripheral proinsulin conversion to insulin and C-peptide is not likely. The basal endogenous secretion rate for proinsulin is 68.7 pmol per hour.

Adult↗
PubMed1979

Proinsulin precursors in catfish pancreatic islets.

The purpose of these experiments was to determine whether insulin-related peptides, larger than proinsulin, could be detected in pancreatic islet cells. Catfish pancreatic islets were incubated with radiolabeled amino acids. After 15- to 60-min incubation, two acid-alcohol-extractable peptides, larger than proinsulin, were detected which were approximately of Mr = 12,000 and 11,000 (12 K and 11K, respectively). They migrated as single polypeptide chains by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis under reducing conditions, and were therefore not aggregates of insulin or proinsulin. The 12 K protein had identical mobility with catfish preoproinsulin synthesized in a wheat germ cell-free system. On standard electrophoresis at pH 8.9, the 12 K protein migrated separately from proinsulin and was at least 65% one protein with two to three minor contaminants. The 12 K and 11 K proteins were chemically related to insulin and proinsulin as shown by tryptic peptide analysis, using cation exchange resin chromatography, and by two-dimensional tryptic peptide maps. Analysis of the tryptic digest of the 12 K protein, compared to proinsulin after leucine aminopeptidase treatment, suggested that the NH2 terminus of the larger protein was different from that of proinsulin. These peptides were specifically bound to anti-insulin antibody. The binding was only 5 to 8% of the protein added, but was specific for the 12 K and 11 K proteins when the immunoprecipitates were examined by electrophoresis and not from contaminating proinsulin. During the continuous incubation of the islets with [3H]leucine, 12 K and 11 K proteins were synthesized in the cell before proinsulin. When islets were first incubated with [3H]leucine for 30 min followed by incubation with excess unlabeled leucine, the 12 K and 11 K proteins appeared to show a precursor-product relationship to proinsulin and insulin. Even when total islet protein synthesis was inhibited by cycloheximide (100 microgram/ml), proinsulin continued to be synthesized for up to 2 h. This suggested that the conversion of the proinsulin precursors to proinsulin in the fish is a post-translational event.

Animals↗
PubMed1970

Human serum proinsulin.

Gel filtration of human serum extracts on Bio-Gel P-30 columns produced two peaks of material reactive with insulin antisera. The earlier eluting fraction appeared at the elution position of proinsulin (serum proinsulin-like component, PLC) while the second fraction corresponded in elution volume to insulin. In assays using porcine insulin-(131)I and an antiserum against porcine insulin, human pancreatic proinsulin was less reactive than human insulin. Serial dilutions of the serum PLC in the immunoassay showed immunological identity with the human proinsulin standard. Partial tryptic digestion of the serum PLC yielded products with increased immunological reactivity as estimated with insulin as the standard. With larger amounts of trypsin, all the serum PLC was converted to insulin-like components (desthreonine and desoctapeptide insulin). On the basis of these results we conclude that the earlier eluting fraction of human serum extracts is proinsulin. The fasting values of proinsulin in normal subjects ranged between 0.05 and 0.4 ng/ml, representing from 5 to 48% of the insulin concentration. In one subject the values of proinsulin were higher than those of insulin. After oral administration of 100 g of glucose, the proinsulin levels tended to rise similarly to insulin. Three obese patients with hyperinsulinemia had higher fasting levels of proinsulin and a greater increase after glucose than the normal subjects. As the high levels of proinsulin coexisted with raised insulin concentration in these obese subjects, the relative proportions of the two hormones were in the same range observed in the normal group. Thus hyperinsulinemia in these obese subjects was not accompanied by an increase in the fraction of serum proinsulin. When the values for serum proinsulin were expressed as percentage of the insulin levels, there was a decrease in the per cent proinsulin in the first hour of the glucose tolerance test. After the second hour, the per cent tended to rise towards the fasting levels.

Adolescent↗