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Diabetes Apr 1994Pancreatic beta-cell dysfunction is a characteristic of non-insulin-dependent diabetes mellitus (NIDDM). An aspect of this dysfunction is that an increased proportion of... (Review)
Review
Pancreatic beta-cell dysfunction is a characteristic of non-insulin-dependent diabetes mellitus (NIDDM). An aspect of this dysfunction is that an increased proportion of proinsulin is secreted, but an actual beta-cell defect that leads to hyperproinsulinemia is unknown. Nevertheless, an impairment in beta-cell proinsulin conversion mechanism has been suggested as the most likely cause. Insulin is produced from its precursor molecule, proinsulin, by limited proteolytic cleavage at two dibasic sequences (Arg31, Arg32 and Lys64, Arg65). Two endopeptidase activities catalyze this cleavage: PC2 and PC3. PC2 endopeptidase cleaves predominately at Lys64, Arg65, and PC3 endopeptidase cleaves at Arg31, Arg32. The recent identification and characterization of these endopeptidases has enabled a better understanding of the human proinsulin-processing mechanism. In particular, experimental evidence suggests that the majority of human proinsulin processing is sequential. PC3 cleaves proinsulin first to generate a proinsulin conversion intermediate that is the preferred substrate of PC2. Both PC2 and PC3 activities are influenced by Ca2+ and pH, but the more stringent Ca2+ and pH requirements of PC3 suggest it as the most likely enzyme to regulate proinsulin conversion, as well as initiate it. When an increased demand is placed on the proinsulin-processing mechanism by a glucose-stimulated increase in proinsulin biosynthesis, there is a coordinate increase in PC3 biosynthesis (but not in PC2). This supports PC3 as the key endopeptidase that regulates proinsulin processing. In this perspective, the current concepts of the enzymology and regulation of proinsulin conversion at a molecular level are reviewed.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Amino Acid Sequence; Diabetes Mellitus, Type 2; Endopeptidases; Humans; Islets of Langerhans; Molecular Sequence Data; Proinsulin; Protein Conformation; Protein Processing, Post-Translational
PubMed: 8138054
DOI: 10.2337/diab.43.4.511 -
Diabetes Apr 2016Proinsulin folding within the endoplasmic reticulum (ER) remains incompletely understood, but it is clear that in mutant INS gene-induced diabetes of youth (MIDY),...
Proinsulin folding within the endoplasmic reticulum (ER) remains incompletely understood, but it is clear that in mutant INS gene-induced diabetes of youth (MIDY), progression of the (three) native disulfide bonds of proinsulin becomes derailed, causing insulin deficiency, β-cell ER stress, and onset of diabetes. Herein, we have undertaken a molecular dissection of proinsulin disulfide bond formation, using bioengineered proinsulins that can form only two (or even only one) of the native proinsulin disulfide bonds. In the absence of preexisting proinsulin disulfide pairing, Cys(B19)-Cys(A20) (a major determinant of ER stress response activation and proinsulin stability) preferentially initiates B-A chain disulfide bond formation, whereas Cys(B7)-Cys(A7) can initiate only under oxidizing conditions beyond that existing within the ER of β-cells. Interestingly, formation of these two "interchain" disulfide bonds demonstrates cooperativity, and together, they are sufficient to confer intracellular transport competence to proinsulin. The three most common proinsulin disulfide mispairings in the ER appear to involve Cys(A11)-Cys(A20), Cys(A7)-Cys(A20), and Cys(B19)-Cys(A11), each disrupting the critical Cys(B19)-Cys(A20) pairing. MIDY mutations inhibit Cys(B19)-Cys(A20) formation, but treatment to force oxidation of this disulfide bond improves folding and results in a small but detectable increase of proinsulin export. These data suggest possible therapeutic avenues to ameliorate ER stress and diabetes.
Topics: Animals; Cells, Cultured; Diabetes Mellitus; Disulfides; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; HEK293 Cells; Humans; Insulin; Insulin-Secreting Cells; Mice; Oxidation-Reduction; Oxidative Stress; Proinsulin; Protein Folding
PubMed: 26822090
DOI: 10.2337/db15-1345 -
Frontiers in Endocrinology 2022Toxic misfolding of proinsulin variants in β-cells defines a monogenic diabetes syndrome, designated (MIDY). In our first study (previous article in this issue), we...
Toxic misfolding of proinsulin variants in β-cells defines a monogenic diabetes syndrome, designated (MIDY). In our first study (previous article in this issue), we described a one-disulfide peptide model of a proinsulin folding intermediate and its use to study such variants. The mutations (Leu→Pro, Leu→Pro, and Phe→Ser) probe residues conserved among vertebrate insulins. In this companion study, we describe H and H-C NMR studies of the peptides; key NMR resonance assignments were verified by synthetic C-labeling. Parent spectra retain nativelike features in the neighborhood of the single disulfide bridge (cystine B19-A20), including secondary NMR chemical shifts and nonlocal nuclear Overhauser effects. This partial fold engages wild-type side chains Leu, Leu and Phe at the nexus of nativelike α-helices α and α (as defined in native proinsulin) and flanking β-strand (residues B24-B26). The variant peptides exhibit successive structural perturbations in order: parent (most organized) > Ser >> Pro > Pro (least organized). The same order pertains to (a) overall α-helix content as probed by circular dichroism, (b) synthetic yields of corresponding three-disulfide insulin analogs, and (c) ER stress induced in cell culture by corresponding mutant proinsulins. These findings suggest that this and related peptide models will provide a general platform for classification of MIDY mutations based on molecular mechanisms by which nascent disulfide pairing is impaired. We propose that the syndrome's variable phenotypic spectrum-onsets ranging from the neonatal period to later in childhood or adolescence-reflects structural features of respective folding intermediates.
Topics: Adolescent; Diabetes Mellitus; Disulfides; Humans; Infant, Newborn; Insulin; Proinsulin; Protein Folding
PubMed: 35299958
DOI: 10.3389/fendo.2022.821091 -
Molecular and Cellular Endocrinology Dec 2020Insulin gene mutation is the second most common cause of neonatal diabetes (NDM). It is also one of the genes involved in maturity-onset diabetes of the young (MODY). We...
Insulin gene mutation is the second most common cause of neonatal diabetes (NDM). It is also one of the genes involved in maturity-onset diabetes of the young (MODY). We aim to investigate molecular behaviors of different INS gene variants that may correlate with the clinical spectrum of diabetes phenotypes. In this study, we concentrated on two previously uncharacterized MODY-causing mutants, proinsulin-p.Gly44Arg [G(B20)R] and p.Pro52Leu [P(B28)L] (a novel mutant identified in one French family), and an NDM causing proinsulin-p.(Cys96Tyr) [C(A7)Y]. We find that these proinsulin mutants exhibit impaired oxidative folding in the endoplasmic reticulum (ER) with blocked ER export, ER stress, and apoptosis. Importantly, the proinsulin mutants formed abnormal intermolecular disulfide bonds that not only involved the mutant proinsulin, but also the co-expressed WT-proinsulin, forming misfolded disulfide-linked proinsulin complexes. This impaired the intracellular trafficking of WT-proinsulin and limited the production of bioactive mature insulin. Notably, although all three mutants presented with similar defects in folding, trafficking, and dominant negative behavior, the degrees of these defects appeared to be different. Specifically, compared to MODY mutants G(B20)R and P(B28)L that partially affected folding and trafficking of co-expressed WT-proinsulin, the NDM mutant C(A7)Y resulted in an almost complete blockade of the ER export of WT-proinsulin, decreasing insulin production, inducing more severe ER stress and apoptosis. We thus demonstrate that differences in cell biological behaviors among different proinsulin mutants correlate with the spectrum of diabetes phenotypes caused by the different INS gene mutations.
Topics: Adolescent; Adult; Animals; Cells, Cultured; Diabetes Mellitus, Type 2; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Female; Genetic Association Studies; Genetic Testing; HEK293 Cells; Humans; Insulin; Insulin-Secreting Cells; Male; Mutation; Phenotype; Proinsulin; Protein Folding; Rats
PubMed: 32916194
DOI: 10.1016/j.mce.2020.111025 -
Danish Medical Bulletin Sep 1999
Review
Topics: Animals; Diabetes Mellitus, Type 1; Family Health; Humans; Hyperglycemia; Proinsulin
PubMed: 10514938
DOI: No ID Found -
Acta Diabetologica Latina 1972
Review
Topics: Amino Acids; Animals; Cattle; Humans; Insulin; Islets of Langerhans; Peptides; Proinsulin; Sheep
PubMed: 4563209
DOI: 10.1007/BF01564538 -
Biological Chemistry Nov 2005A single mutation (C96Y) in the Ins2 gene, which disrupts the A7-B7 disulfide bond, causes the diabetic phenotype in Akita mice. We biochemically analyzed the...
A single mutation (C96Y) in the Ins2 gene, which disrupts the A7-B7 disulfide bond, causes the diabetic phenotype in Akita mice. We biochemically analyzed the conformation of wild-type and Akita mutant recombinant proinsulins. Gel filtration chromatography and dynamic light scattering revealed that the apparent size of the mutant proinsulin molecules was significantly larger than that of wild-type proinsulin, even in the absence of intermolecular disulfide bonds. Titration with a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate, demonstrated that the mutant proinsulin was more hydrophobic than the wild type. In addition, circular dichroism studies revealed that the conformation of the mutant proinsulin was less stable than the wild type, which is consistent with the observation that hydrophobic residues are exposed on the surface of the proinsulin molecules. Studies with antiserum against the C-peptide of proinsulin indicated that the mutant proinsulin had an immunoreactivity that was at least one-tenth weaker than wild-type proinsulin, suggesting that the C-peptide of mutant proinsulin is buried inside the aggregate of the proinsulin molecule. These findings indicate that increased hydrophobicity of mutant proinsulin facilitates aggregate formation, providing a clue to the dominant negative effect in the Akita mouse.
Topics: Anilino Naphthalenesulfonates; Animals; Circular Dichroism; Diabetes Mellitus, Experimental; Disulfides; Genes, Dominant; Hydrophobic and Hydrophilic Interactions; Light; Mice; Mice, Transgenic; Molecular Conformation; Mutation; Particle Size; Proinsulin; Recombinant Proteins; Scattering, Radiation
PubMed: 16307473
DOI: 10.1515/BC.2005.124 -
Reviews in Endocrine & Metabolic... Aug 2005
Topics: Animals; Chick Embryo; Embryo, Mammalian; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Insulin; Proinsulin; Receptor, Insulin; Signal Transduction
PubMed: 16151625
DOI: 10.1007/s11154-005-3052-x -
Biotechnology Journal Mar 2024The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk.
BACKGROUND
The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk.
METHODS AND RESULTS
Pseudo-lentivirus containing the bovine β-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein.
CONCLUSION
The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.
Topics: Female; Pregnancy; Animals; Cattle; Humans; Animals, Genetically Modified; Proinsulin; Milk; Recombinant Proteins; Insulin; Peptide Hydrolases
PubMed: 38472101
DOI: 10.1002/biot.202300307 -
Metabolism: Clinical and Experimental Oct 1995Associations between loss of glucose tolerance, insulin resistance, and ischemic heart disease (IHD) are of great current concern. Considerable controversy and... (Review)
Review
Associations between loss of glucose tolerance, insulin resistance, and ischemic heart disease (IHD) are of great current concern. Considerable controversy and uncertainty relates to the mechanism(s) that underlies these associations. Whilst there is some evidence in prospective studies of an association between hyperinsulinemia and future IHD, it is by no means strong or consistent between different studies. Hypertriglyceridemia is another possible factor involved in the linkage between glucose intolerance and IHD. There is good evidence for an affect of plasma nonesterified fatty acids (NEFA) to increase hepatic output of VLDL. Insulin, contrary to some suggestions, acts to lower plasma VLDL by actions directly on hepatic output and activation of adipose tissue lipoprotein lipase, and indirectly via the hormones affect of lowering plasma NEFA. Glycosylation and oxidation of lipoproteins may enhance their atherogenic potential. It is highly probable that procoagulant changes are also important processes predisposing to IHD. Associations between plasminogen activator inhibitor-1 and insulin, intact and 32,33 split proinsulin hypertriglyceridemia, and insulin resistance have been reported, but a unifying hypothesis explaining these links remains elusive. Epidemiological studies now repeated in a number of centers have shown links between infant mortality and birth weight and risk of IHD, and between birth weight and risk of impaired glucose tolerance and non-insulin-dependent diabetes mellitus (NIDDM). It has been proposed, therefore, that impairment of fetal and infant growth may underlie the associations between loss of glucose tolerance and risk of IHD. Animal models form the basis of much current research to test this concept.
Topics: Diabetic Angiopathies; Humans; Insulin; Insulin Resistance; Models, Biological; Proinsulin
PubMed: 7476316
DOI: 10.1016/0026-0495(95)90225-2