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Journal of Biotechnology Feb 2022With the rapid spread of diabetes in human society, the demand for insulin and its precursor (proinsulin) continues to rise. Therefore, the introduction of new methods...
With the rapid spread of diabetes in human society, the demand for insulin and its precursor (proinsulin) continues to rise. Therefore, the introduction of new methods for their production is essential. In the present study, human proinsulin, while ligated to αB-crystallin chaperone, was effectively expressed in the prokaryotic host system and then purified by the ion-exchange chromatography at high purity (>97%). In the next step, human proinsulin with relatively high efficiency was released chemically from the hybrid protein (αB-pIns) and then purified using an appropriate gel filtration column. The SDS-PAGE and HPLC analyses confirmed the high purity, while mass spectroscopy assessment verified the exact molecular mass of the human proinsulin. Using a well-established protocol, the protein was folded in a one-step folding process with a yield of about 70%. The assessment of the secondary structures of the human proinsulin by Raman and FTIR spectroscopy suggested that this protein is rich in α-helix. Also, the conformation of disulfide bonds in the folded proinsulin was confirmed by Raman spectroscopy. The recombinant human proinsulin also demonstrated hypoglycemic activity and mitogenic action (induction of cell proliferation). The method proposed in this work for the production of human proinsulin is easy to run and does not depend on expensive and complex equipment. Thus, it can be used in the industrial production of human proinsulin.
Topics: Chromatography, Gel; Humans; Insulin; Molecular Chaperones; Proinsulin; Protein Structure, Secondary
PubMed: 35066065
DOI: 10.1016/j.jbiotec.2022.01.002 -
CRC Critical Reviews in Biochemistry Feb 1972
Review
Topics: Allergy and Immunology; Animals; Cattle; Chemical Phenomena; Chemistry; Glucose; Humans; Hydrolysis; Insulin; Insulin Antibodies; Kinetics; Molecular Weight; Oxidation-Reduction; Pancreas; Peptide Hydrolases; Peptides; Proinsulin; Swine; Trypsin
PubMed: 4570578
DOI: 10.3109/10409237209102544 -
Diabetologia Jun 2020It is unclear whether type 1 diabetes is a single disease or if endotypes exist. Our aim was to use a unique collection of pancreas samples recovered soon after disease...
AIMS/HYPOTHESIS
It is unclear whether type 1 diabetes is a single disease or if endotypes exist. Our aim was to use a unique collection of pancreas samples recovered soon after disease onset to resolve this issue.
METHODS
Immunohistological analysis was used to determine the distribution of proinsulin and insulin in the islets of pancreas samples recovered soon after type 1 diabetes onset (<2 years) from young people diagnosed at age <7 years, 7-12 years and ≥13 years. The patterns were correlated with the insulitis profiles in the inflamed islets of the same groups of individuals. C-peptide levels and the proinsulin:C-peptide ratio were measured in the circulation of a cohort of living patients with longer duration of disease but who were diagnosed in these same age ranges.
RESULTS
Distinct patterns of proinsulin localisation were seen in the islets of people with recent-onset type 1 diabetes, which differed markedly between children diagnosed at <7 years and those diagnosed at ≥13 years. Proinsulin processing was aberrant in most residual insulin-containing islets of the younger group but this was much less evident in the group ≥13 years (p < 0.0001). Among all individuals (including children in the middle [7-12 years] range) aberrant proinsulin processing correlated with the assigned immune cell profiles defined by analysis of the lymphocyte composition of islet infiltrates. C-peptide levels were much lower in individuals diagnosed at <7 years than in those diagnosed at ≥13 years (median <3 pmol/l, IQR <3 to <3 vs 34.5 pmol/l, IQR <3-151; p < 0.0001), while the median proinsulin:C-peptide ratio was increased in those with age of onset <7 years compared with people diagnosed aged ≥13 years (0.18, IQR 0.10-0.31) vs 0.01, IQR 0.009-0.10 pmol/l; p < 0.0001).
CONCLUSIONS/INTERPRETATION
Among those with type 1 diabetes diagnosed under the age of 30 years, there are histologically distinct endotypes that correlate with age at diagnosis. Recognition of such differences should inform the design of future immunotherapeutic interventions designed to arrest disease progression.
Topics: Adolescent; Age Factors; Child; Diabetes Mellitus, Type 1; Humans; Insulin; Male; Pancreas; Proinsulin
PubMed: 32172310
DOI: 10.1007/s00125-020-05115-6 -
Diabetologia Apr 1996Proinsulin and insulin binding in IM-9 lymphoblasts show curvilinear Scatchard plots, which may be explained by two binding sites, negative cooperativity of receptors,...
Proinsulin and insulin binding in IM-9 lymphoblasts show curvilinear Scatchard plots, which may be explained by two binding sites, negative cooperativity of receptors, or both. Using flow-cytometric analysis of insulin binding, we were able to distinguish and separate two different IM-9 cell fractions. In both fractions, Scatchard plots for specific binding of insulin and proinsulin were linear, suggesting the presence of two distinct populations of receptors. Type 1 cells showed low capacity but high affinity of insulin binding (16,300 +/- 3,000 sites/cell; Kd 0.4 +/- 0.1 nmol/l). Proinsulin and insulin-like growth factor 1 (IGF-1) were significantly less potent in competition. MA-20, a specific antibody against human insulin receptors, inhibited insulin binding by 80%, while the specific antibody against human IGF-1 receptors, alpha IR-3, had no effect. Pretreatment with insulin decreased insulin binding by 90%. 125I-insulin displayed stepwise dissociation with the rate markedly enhanced by cold insulin. Type 2 cells exhibited significantly different binding characteristics with higher capacity but lower affinity of 125I-insulin binding (430,000 +/- 25,000 sites/cell, p < 0.001 vs type 1; Kd 2 +/- 0.4 nmol/l, p < 0.02 vs type 1). Proinsulin competed with similar potency for insulin binding, while IGF-1 was still less potent. 125I-proinsulin showed a significantly higher binding affinity than 125I-insulin (Kd 0.5 +/- 0.3 nmol/l, p < 0.05) with 50,000 +/- 10,000 binding sites/cell. C-peptide was able to compete for 125I-proinsulin, but not for 125I-insulin binding. MA-20 did not influence 125I-proinsulin binding, but inhibited 125I-insulin binding by 50%, whereas alpha IR-3 increased proinsulin binding 1.5-fold with no effect on insulin binding. Preincubation with insulin decreased insulin binding by 50% and proinsulin binding by 10%. The dissociation of 125I-proinsulin showed linear first-order kinetics and was not significantly accelerated by cold proinsulin. Furthermore, the tyrosine phosphorylation of a 65 kDa protein was stimulated to a significantly greater extent by proinsulin than by insulin, indicating activation of different signalling cascades. DNA analysis revealed that type 1 cells were predominantly in the G1 phase, whereas type 2 cells were in the S and G2 + M phases of the cell cycle. We conclude, that IM-9 lymphoblasts were separated by flow-cytometry into one fraction with typical insulin receptors and a second fraction with high affinity binding sites for proinsulin. High affinity proinsulin binding sites were distinguished from typical insulin receptors by: 1) higher affinity for proinsulin than insulin, 2) inhibition of proinsulin binding by C-peptide but not by the insulin receptor antibody MA-20, 3) non-co-operative first order dissociation kinetics of proinsulin binding, 4) resistance to down-regulation by insulin, and 5) differences in signal transduction.
Topics: Antibodies, Monoclonal; Antibody Specificity; Binding Sites; Binding, Competitive; Cell Line; Flow Cytometry; Humans; Insulin; Insulin-Like Growth Factor I; Iodine Radioisotopes; Kinetics; Lymphocytes; Proinsulin; Receptor, Insulin
PubMed: 8777991
DOI: 10.1007/BF00400673 -
Biochemistry and Molecular Biology... Jan 1995A fusion gene encoding double-C-peptide human proinsulin was constructed by insertion of a DNA fragment encoding human C-peptide into the 5'-terminal C-peptide coding...
A fusion gene encoding double-C-peptide human proinsulin was constructed by insertion of a DNA fragment encoding human C-peptide into the 5'-terminal C-peptide coding sequence of a synthetic human proinsulin gene with correct reading frame and over-expressed in E. coli. The purified double-C-peptide human proinsulin shows decreased activity in receptor binding and insulin immune assays as compared with human proinsulin. Disulphide bond reconstitution studies demonstrate that there is not much more influence of the protein concentration on the yield of refolded double-C-peptide human proinsulin. The double-C-peptide human proinsulin shows a 1.86-fold human C-peptide immune activity as compared with that of human proinsulin and gives a good yield of the molecule with correct disulphide bonds in reconstitution studies strongly suggesting the existence of very flexible conformation of the C-peptide.
Topics: Amino Acid Sequence; Base Sequence; C-Peptide; Cloning, Molecular; Escherichia coli; Gene Expression Regulation, Bacterial; Humans; Insulin; Molecular Sequence Data; Proinsulin; Protein Binding; Recombinant Proteins; Sulfur
PubMed: 7735138
DOI: No ID Found -
Diabetes Aug 2011Endoplasmic reticulum (ER) stress has been described in pancreatic β-cells after onset of diabetes-a situation in which failing β-cells have exhausted available...
OBJECTIVE
Endoplasmic reticulum (ER) stress has been described in pancreatic β-cells after onset of diabetes-a situation in which failing β-cells have exhausted available compensatory mechanisms. Herein we have compared two mouse models expressing equally small amounts of transgenic proinsulin in pancreatic β-cells.
RESEARCH DESIGN AND METHODS
In hProCpepGFP mice, human proinsulin (tagged with green fluorescent protein [GFP] within the connecting [C]-peptide) is folded in the ER, exported, converted to human insulin, and secreted. In hProC(A7)Y-CpepGFP mice, misfolding of transgenic mutant proinsulin causes its retention in the ER. Analysis of neonatal pancreas in both transgenic animals shows each β-cell stained positively for endogenous insulin and transgenic protein.
RESULTS
At this transgene expression level, most male hProC(A7)Y-CpepGFP mice do not develop frank diabetes, yet the misfolded proinsulin perturbs insulin production from endogenous proinsulin and activates ER stress response. In nondiabetic adult hProC(A7)Y-CpepGFP males, all β-cells continue to abundantly express transgene mRNA. Remarkably, however, a subset of β-cells in each islet becomes largely devoid of endogenous insulin, with some of these cells accumulating large quantities of misfolded mutant proinsulin, whereas another subset of β-cells has much less accumulated misfolded mutant proinsulin, with some of these cells containing abundant endogenous insulin.
CONCLUSIONS
The results indicate a source of pancreatic compensation before the development of diabetes caused by proinsulin misfolding with ER stress, i.e., the existence of an important subset of β-cells with relatively limited accumulation of misfolded proinsulin protein and maintenance of endogenous insulin production. Generation and maintenance of such a subset of β-cells may have implications in the avoidance of type 2 diabetes.
Topics: Animals; Endoplasmic Reticulum; Female; Humans; Islets of Langerhans; Male; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Proinsulin; Protein Folding; Transgenes
PubMed: 21677281
DOI: 10.2337/db10-1671 -
The Journal of Clinical Investigation May 1973The renal extraction and excretion of bovine proinsulin, insulin, and C-peptide and the contribution of the kidney to their total metabolic clearance rate (MCR) were...
The renal extraction and excretion of bovine proinsulin, insulin, and C-peptide and the contribution of the kidney to their total metabolic clearance rate (MCR) were studied in the rat. Metabolic clearance rates were measured by the constant infusion technique and plasma and urine concentrations of each polypeptide were determined by radioimmunoassay. The MCR of insulin (16.4+/-0.4 ml/min) was significantly greater than that of either proinsulin (6.7+/-0.3 ml/min) or C-peptide (4.6+/-0.2 ml/min). Metabolic clearance rates were independent of plasma levels over a range of steady-state plasma concentrations varying from 1 to 15 ng/ml.In contrast to the differences in their metabolic clearance rates, the renal disposition of the three polypeptides was similar, being characterized by high extraction and very low urinary clearance. The renal arteriovenous difference of proinsulin, insulin, and C-peptide averaged 36, 40, and 44%, respectively, and was linearly related to their arterial concentration between 2 and 25 ng/ml. When glomerular filtration was markedly reduced or stopped by ureteral obstruction, the renal extraction of proinsulin, insulin, and C-peptide was invariably greater than the simultaneously measured extraction of inulin, indicating that these polypeptides are removed from the renal circulation by both glomerular filtration and direct uptake from peritubular capillary blood. The fractional urinary clearance of each polypeptide never exceeded 0.6%, indicating that more than 99% of the amount filtered was sequestered in the kidney. The renal removal of proinsulin and C-peptide from the circulation accounts for 55 and 69% of their metabolic clerance rates, while the renal contribution to the peripheral metabolism of insulin was smaller, averaging 33%. This difference is due to the fact that insulin, but not the other two polypeptides, is metabolized to a significant extent by the liver. These results define the renal handling of proinsulin, insulin, and C-peptide in the rat and indicate that in this species the kidney represents a major site for insulin metabolism and is the main organ responsible for the degradation of proinsulin and C-peptide.
Topics: Animals; Chromatography, Gel; Glomerular Filtration Rate; Insulin; Kidney; Kidney Concentrating Ability; Kidney Glomerulus; Male; Metabolic Clearance Rate; Peptides; Proinsulin; Radioimmunoassay; Rats; Regional Blood Flow
PubMed: 4700486
DOI: 10.1172/JCI107277 -
Chemical Communications (Cambridge,... Nov 2010A convergent synthetic strategy based on modern chemical ligation methods was used to make human proinsulin. The synthetic protein was characterized by LCMS, CD...
A convergent synthetic strategy based on modern chemical ligation methods was used to make human proinsulin. The synthetic protein was characterized by LCMS, CD spectroscopy, and by 1D- and 2D-NMR spectroscopy. Synthetic human proinsulin had full biochemical activity in a receptor-binding assay.
Topics: Amino Acid Sequence; Circular Dichroism; Humans; Molecular Sequence Data; Proinsulin; Stereoisomerism
PubMed: 20877850
DOI: 10.1039/c0cc03141k -
Clinical Chemistry Jun 2021Determination of C-peptide is important in the investigation of unexplained hyperinsulinemic hypoglycemia because a high C-peptide concentration usually indicates...
BACKGROUND
Determination of C-peptide is important in the investigation of unexplained hyperinsulinemic hypoglycemia because a high C-peptide concentration usually indicates endogenous insulin hypersecretion. Insulin autoimmune syndrome (IAS) denotes hyperinsulinemic hypoglycemia due to insulin-binding antibodies that prolong insulin half-life. C-peptide clearance is considered to be unaffected, and although a marked C-peptide immunoreactivity in hypoglycemic samples has been reported, it has been suspected to be artifactual. High-resolution mass spectrometry enables examination of the basis of C-peptide-immunoreactivity in IAS.
METHODS
Precipitation of plasma with polyethylene glycol was followed by C-peptide immunoassay. Plasma peptides extracted by solvent precipitation were characterized by nano-LC-MS/MS and analyzed using an untargeted data-dependent method. Peptides related to proinsulin, in amino acid sequence, were identified using proprietary bioinformatics software and confirmed by repeat LC-MS/MS analysis. Gel filtration chromatography coupled to LC-MS/MS was used to identify proinsulin-related peptides present in IAS immunocomplexes. Results were compared with those from C-peptide immunoassay.
RESULTS
Polyethylene glycol precipitation of IAS plasma, but not control plasma, depleted C-peptide immunoreactivity consistent with immunoglobulin-bound C-peptide immunoreactivity. LC-MS/MS detected proinsulin and des 31,32 proinsulin at higher abundance in IAS plasma compared with control plasma. Analysis by gel filtration chromatography coupled to LC-MS/MS demonstrated proinsulin and des 31,32 proinsulin, but no C-peptide, in plasma immunocomplexes.
CONCLUSIONS
Antibody binding can enrich proinsulin and des 31,32 proinsulin in IAS immunocomplexes. Proinsulin cross-reactivity in some C-peptide immunoassays can lead to artifactually increased C-peptide results.
Topics: Autoimmune Diseases; C-Peptide; Chromatography, Liquid; Humans; Hyperinsulinism; Hypoglycemia; Insulin; Insulin Antibodies; Molecular Weight; Peptides; Polyethylene Glycols; Proinsulin; Tandem Mass Spectrometry
PubMed: 34051096
DOI: 10.1093/clinchem/hvab043 -
Diabetes Feb 1984The binding and biologic properties of human proinsulin produced by recombinant DNA technology have been determined. The biosynthetic human proinsulin was iodinated...
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)
Topics: Animals; Binding, Competitive; Deoxyglucose; Glucose; Humans; Insulin; Kinetics; Proinsulin; Rats; Rats, Inbred Strains
PubMed: 6363165
DOI: 10.2337/diab.33.2.111