-
Biochemical and Biophysical Research... Jul 2011Disproportionate hyperproinsulinemia is an indicator of β-cell dysfunction in diabetes and the basis underlying this abnormality remains obscure. Recently, we have...
Disproportionate hyperproinsulinemia is an indicator of β-cell dysfunction in diabetes and the basis underlying this abnormality remains obscure. Recently, we have found proinsulin is an aggregation-prone molecule inherent with a low relative folding rate and maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO) in normal β-cells as a result of the integration of maturation and disposal processes. PIHO is susceptible to environmental and genetic influences. Perturbation of PIHO produces a number of toxic consequences with known association to β-cell failure in diabetes. To explore whether the perturbation of PIHO has a link to disproportionate hyperproinsulinemia, we investigated proinsulin conversion and the involved prohormone convertase 1/3 (PC1/3) and 2 (PC2) in mouse Ins2(+/Akita) islets/β-cells that preserve a primary PIHO disorder due to a mutation (C96Y) in the insulin 2 (Ins2) gene. Our metabolic-labeling studies found an increased ratio of proinsulin to insulin in the cellular or released proteins of Ins2(+/Akita) islets. Histological, metabolic-labeling, and RT-PCR analyses revealed decreases of the PC1/3 and PC2 immunoreactivities in the β-cells of Ins2(+/Akita) islets in spite of no declines of these two convertases at the transcriptional and translational levels. Immunoblot analyses in cloned Ins2(+/Akita) β-cells further confirmed the increased ratio of proinsulin to insulin despite the levels of PC1/3 and PC2 proteins were not reduced somehow. The findings demonstrate that the perturbation of PIHO results in defects in the subsequent conversion process of proinsulin and is a contributor to the occurrence of disproportionate hyperproinsulinemia in diabetes.
Topics: Animals; Hyperinsulinism; Insulin; Insulin-Secreting Cells; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Proinsulin
PubMed: 21723250
DOI: 10.1016/j.bbrc.2011.06.119 -
Diabetes Jan 1993The plasma ratio of proinsulin/insulin is raised in people with NIDDM. A relative hypersecretion of proinsulin is thought to be the cause, because pancreas extracts from...
The plasma ratio of proinsulin/insulin is raised in people with NIDDM. A relative hypersecretion of proinsulin is thought to be the cause, because pancreas extracts from diabetic rats have a raised proinsulin/insulin ratio. We tested the hypothesis that the pancreatic proinsulin/insulin mismatch results from hyperglycemia-induced beta-cell degranulation. Normal rats made hyperglycemic with 48-h glucose infusions had a raised pancreatic percentage of proinsulin. In contrast, rats infused with enough glucose to induce compensatory hyperinsulinemia without changing the plasma glucose level had a normal percentage of proinsulin. The raised percentage of proinsulin in the hyperglycemic rats reflected a reduction in pancreatic insulin content. Administering an inhibitor of insulin release, diazoxide, to hyperglycemic rats blocked the fall in pancreatic insulin content and prevented the rise in the percentage of proinsulin. Normal rats infused with tolbutamide for 3 days and enough glucose to maintain euglycemia had a 50% reduction in pancreatic insulin content. The beta-cell degranulation from this nonhyperglycemic mechanism resulted in a raised pancreatic percentage of proinsulin. In summary, chronic hyperglycemia causes beta-cell degranulation primarily because of hyperstimulated insulin release. The net result is a rise in the ratio of immature (proinsulin-rich) to mature (insulin-rich) granules, which is reflected as an increased relative proportion of proinsulin. Mobilization of these proinsulin-enriched granules may explain the relative hypersecretion of proinsulin that occurs with diabetes.
Topics: Animals; Blood Glucose; Chromatography, High Pressure Liquid; Hyperglycemia; Insulin; Male; Pancreas; Proinsulin; Rats; Rats, Sprague-Dawley; Tolbutamide
PubMed: 8420818
DOI: 10.2337/diab.42.1.22 -
The Journal of Clinical Investigation Apr 1972The removal of bovine proinsulin by the isolated perfused rat liver has been studied and the results compared with the removal of insulin. At high concentrations of...
The removal of bovine proinsulin by the isolated perfused rat liver has been studied and the results compared with the removal of insulin. At high concentrations of insulin (> 180 ng/ml) the removal process was saturated and the t(1/2) varied between 35 and 56 min. With low initial insulin levels the disappearance followed first-order kinetics, the mean regression coefficient being - 0.022, t(1/2) 13.8 min, and the hepatic extraction 4.0 ml/min. The results with proinsulin were in striking contrast to these findings. At both high and low concentrations the hepatic removal of proinsulin was considerably slower, averaging 10-15 times less than that of insulin. Specific immunoassay techniques and gel filtration of samples taken from perfusions to which both labeled and unlabeled proinsulin had been added did not show conversion to either insulin or the C-peptide. Bovine and rat (131)I-labeled proinsulins were degraded more slowly than bovine insulin-(131)I by bovine and rat liver homogenates. Both proinsulin and insulin inhibited the degradation of insulin-(131)I, equimolar quantities of proinsulin being 2-5 times less effective than insulin. These results indicate significant differences in the capacity of the liver to remove and degrade insulin and proinsulin. The low hepatic extraction of proinsulin may account for its prolonged half-life in vivo and contribute to its relatively high plasma concentration in the fasting state. Furthermore this finding will have to be taken into account in the interpretation of changes in the proinsulin:insulin ratios in peripheral blood in a variety of metabolich situations.
Topics: Animals; Chromatography, Gel; Half-Life; Immunoassay; In Vitro Techniques; Insulin; Iodine Isotopes; Kinetics; Liver; Male; Perfusion; Proinsulin; Rats
PubMed: 5014618
DOI: 10.1172/JCI106886 -
Proceedings of the National Academy of... Sep 1992Experiments using recombinant vaccinia viruses expressing rat proinsulin I coinfected into COS-7 cells with recombinant vaccinia virus expressing human furin, human PC2,...
Experiments using recombinant vaccinia viruses expressing rat proinsulin I coinfected into COS-7 cells with recombinant vaccinia virus expressing human furin, human PC2, mouse PC3 (subtilisin-related proprotein convertases 1-3, respectively), or yeast Kex2 indicate that in this system both Kex2 and furin produce mature insulin, whereas PC2 selectively cleaves proinsulin at the C-peptide-A-chain junction. This is a property consistent with its probable identity with the rat insulinoma granule type II proinsulin processing activity as described by Davidson et al. [Davidson, H. W., Rhodes, C. J. & Hutton, J. C. (1988) Nature (London) 333, 93-96]. PC3 generates mature insulin but cleaves preferentially at the proinsulin B-chain-C-peptide junction. This pattern of cleavage by PC3 is similar, but not identical, to that of the highly B-chain-C-peptide junction-selective type I activity as described by Davidson et al., perhaps due to the presence of a P4 arginine residue near the C-peptide-A-chain junction unique to the rat proinsulins. These results along with data presented on the expression of both PC2 and PC3 in islet beta cells strongly support the conclusion that these proteases are involved in the conversion of proinsulin to insulin in vivo.
Topics: Amino Acid Sequence; Animals; Furin; Humans; Immunohistochemistry; Insulinoma; Islets of Langerhans; Molecular Sequence Data; Molecular Weight; Proinsulin; Proprotein Convertase 2; Proprotein Convertases; Protein Processing, Post-Translational; Rats; Serine Endopeptidases; Substrate Specificity; Subtilisins
PubMed: 1528899
DOI: 10.1073/pnas.89.18.8822 -
Trends in Neurosciences Mar 1995The multifunctional cytokines of the family of insulin and insulin-like growth factors (IGFs) have not yet gained general recognition as essential cell signals for the... (Review)
Review
The multifunctional cytokines of the family of insulin and insulin-like growth factors (IGFs) have not yet gained general recognition as essential cell signals for the development of the vertebrate nervous system. This is, in part, a consequence of previous constraints in our thinking, focused for many years on the endocrine roles of these factors in late mammalian development and postnatal stages. The cellular distribution of the components of the insulin and IGFs signalling system in the developing mammalian and avian CNS is remarkably conserved. While receptors are widespread, the much less abundant factors and modulatory proteins are highly regulated in time and space. Progression of neural development through the steps of cell proliferation, differentiation, maturation and survival is stimulated, at least in culture, by proinsulin and insulin and the IGFs. Thus, these factors might be important autocrine and paracrine signals during development of the CNS.
Topics: Animals; Central Nervous System; Insulin; Proinsulin; Somatomedins
PubMed: 7754526
DOI: 10.1016/0166-2236(95)93892-2 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Mar 2017Proinsulin (Pins) is the precursor of insulin. The expression of proinsulin in Escherichia coli forms inclusion body, so that the recombinant protein should be processed...
Proinsulin (Pins) is the precursor of insulin. The expression of proinsulin in Escherichia coli forms inclusion body, so that the recombinant protein should be processed with multiple steps to form active insulin. With the development in biotechnology, cell-free protein synthesis (CFPS) system is becoming a valuable tool in protein expression by decoupling the cell growth with protein production, which allows it to express proteins that would interfere with cell physiology. In this study, we synthesized soluble proinsulin in CFPS system in order to establish a new approach for both insulin expression and delivery. The soluble proinsulin was successfully expressed in CFPS system by fusing proinsulin with two types of fluorescent protein. The expression of Pins-mCherry was confirmed by Western blotting analysis, and the Pins-eGFP titer was (12.28±3.45) μg/mL in CFPS system. These results implicated that the proinsulin was expressed partially in soluble form. Here, for the first time, we successfully expressed soluble proinsulin in CFPS system by fluorescent protein fusion. These results provide useful information in developing new insulin expression and delivery method.
Topics: Cell-Free System; Escherichia coli; Green Fluorescent Proteins; Insulin; Luminescent Proteins; Proinsulin; Protein Biosynthesis; Recombinant Fusion Proteins; Red Fluorescent Protein
PubMed: 28941344
DOI: 10.13345/j.cjb.160452 -
Journal of Chromatography. B,... Nov 2022Routine immunoassays for insulin and C-peptide have the potential to cross-react with partially processed proinsulin products, although in healthy patients these are...
Routine immunoassays for insulin and C-peptide have the potential to cross-react with partially processed proinsulin products, although in healthy patients these are present at such low levels that the interference is insignificant. Elevated concentrations of proinsulin and des-31,32 proinsulin arising from pathological conditions, or injected insulin analogues, however can cause significant assay interferences, complicating interpretation. Clinical diagnosis and management therefore sometimes require methods that can distinguish true insulin and C-peptide from partially processed proinsulin or injected insulin analogues. In this scenario, the high specificity of mass spectrometric analysis offers potential benefit for patient care. A high throughput targeted LC-MS/MS method was developed as a fit for purpose investigation of insulin, insulin analogues, C-peptide and proinsulin processing intermediates in plasma samples from different patient groups. Using calibration standards and bovine insulin as an internal standard, absolute concentrations of insulin and C-peptide were quantified across a nominal human plasma postprandial range and correlated strongly with immunoassay-based measurements. The ability to distinguish between insulin, insulin analogues and proinsulin intermediates in a single extraction is an improvement over existing immunological based techniques, offering the advantage of exact identification of the species being measured. The method promises to aid in the detection of circulating peptides which have previously been overlooked but may interfere with standard insulin and C-peptide immunoassays.
Topics: Humans; Cattle; Animals; Proinsulin; C-Peptide; Chromatography, Liquid; Insulin-Secreting Cells; Tandem Mass Spectrometry; Insulin; Peptides
PubMed: 36242807
DOI: 10.1016/j.jchromb.2022.123482 -
Methods in Molecular Biology (Clifton,... 2000
Review
Topics: Animals; C-Peptide; Humans; Insulin; Isotope Labeling; Proinsulin; Spectrometry, Mass, Electrospray Ionization
PubMed: 10948509
DOI: 10.1385/1-59259-045-4:293 -
The Journal of Biological Chemistry Jul 1992Rat hepatoma (FAO) cells were stably transfected with the gene encoding either rat proinsulin II (using the DOL retroviral vector) or human proinsulin (using the RSV...
Rat hepatoma (FAO) cells were stably transfected with the gene encoding either rat proinsulin II (using the DOL retroviral vector) or human proinsulin (using the RSV retroviral vector). Using the DOL vector, production of insulin immunoreactive material was stimulated up to 30-fold by dexamethasone (5 x 10(-7) M). For both proinsulins, fractional release of immunoreactive material relative to cellular content was high, in keeping with the absence of any storage compartment for secretory proteins in these cells. Pulse-chase experiments showed kinetics of release of newly synthesized products in keeping with release via the constitutive pathway. High performance liquid chromatography analysis showed immunoreactivity in the medium distributed between three peaks. For rat proinsulin II, the first coeluted with intact proinsulin; the second coeluted with des-64,65 split proinsulin (the product of endoproteolytic attack between the insulin A-chain and C-peptide followed by trimming of C-terminal basic residues by carboxypeptidase); the third (and minor peak) coeluted with native (fully processed) insulin. For human proinsulin, by contrast, the second peak coeluted with des-31,32 split proinsulin (split and trimmed at the B-chain/C-peptide junction). Analysis of cellular extracts showed intact proinsulin as the major product. The generation of the putative conversion intermediates and insulin was not due to proteolysis of proinsulin after its release but rather to an intracellular event. The data suggest that proinsulin, normally processed in secretory granules and released via the regulated pathway, may also be processed, albeit less efficiently, by the constitutive pathway conversion machinery. The comparison of the sites preferentially cleaved in rat II or human proinsulin suggests cleavage by endoprotease(s) with a preference for R/KXR/KR as substrate.
Topics: Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; DNA; Dexamethasone; Electrophoresis, Gel, Pulsed-Field; Kinetics; Liver Neoplasms, Experimental; Molecular Sequence Data; Precipitin Tests; Proinsulin; Protein Processing, Post-Translational; Radioimmunoassay; Rats; Transfection; Tumor Cells, Cultured
PubMed: 1634510
DOI: No ID Found -
The Journal of the Association of... Mar 1973
Topics: Animals; Humans; Immunoassay; Insulin; Proinsulin; Swine
PubMed: 4799819
DOI: No ID Found