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  • 1
    Electronic Resource
    Electronic Resource
    Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor (2002)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 81 (2002), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The neurotrophin responsive gene vgf is widely expressed in central and peripheral neurones, and in certain neuroendocrine cell populations. Its encoded VGF precursor protein (proVGF1: 617 amino acids in rat, 615 in man, 〉 85% homology) gives rise to several low molecular weight species. We studied a range of neuroendocrine and neuronal cells, in which VGF-processing products were prominent with an apparent molecular weight of 20 and 10 kDa (VGF20 and VGF10, respectively). Such peptides were recognized by antibodies specific for the C-terminal rat VGF nonapeptide, thus indicating that they included the C-terminus of proVGF. Ectopic expression of the neuroendocrine-specific prohormone convertases PC1/3 or PC2 in GH3 cells showed that both could generate VGF20, while VGF10 was preferentially produced by PC1/3. Site-directed mutagenesis was used to identify the KRKRKK488 motif as the target within VGF sequence which leads to the production of VGF20. Molecular characterization of rat VGF10, on the other hand, revealed that this peptide is produced by cleavage at the RPR555 site. By the combined use of high-resolution separation techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry and manual Edman degradation we identified in rat brain a VGF fragment analogous to bovine peptide V and two novel peptides also derived from the C-terminal region of proVGF.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.2002.00842.x
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  • 2
    Electronic Resource
    Electronic Resource
    Substrate specificity of proinsulin conversion in the constitutive pathway of transfected FAO (hepatoma) cells (1993)
    Springer
    Diabetologia 36 (1993), S. 1322-1325 
    Details
    ISSN: 1432-0428
    Keywords: Constitutive secretion ; prohormone processing ; proinsulin ; hepatoma (FAO cell)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Proinsulin is usually targetted to the regulated secretory pathway of beta cells, and converted to insulin in beta granules. Under certain pathological situations, a significant amount of proinsulin becomes diverted to the constitutive pathway. To study the kinetics of proinsulin conversion in the constitutive pathway, FAO (hepatoma) cells, which secrete proteins uniquely via this pathway and not the regulated pathway, were stably transfected with cDNA encoding human, rat I or rat II proinsulin. Products released to the medium of transfected cells were analysed by reversed phase HPLC and radioimmunoassay. For human proinsulin, des 31,32 split proinsulin (the conversion intermediate resulting from cleavage only at the B-chain/C-peptide junction followed by trimming of C-terminal basic residues by carboxypeptidase) was the only detectable conversion intermediate; for rat proinsulin II it was des 64,65 split proinsulin (cleaved and trimmed only at the C-peptide/A-chain junction); for rat proinsulin I, both intermediates were seen. Complete processing to insulin occurred for all three, but was most extensive for rat proinsulin I. When considered with the corresponding proinsulin sequences, these data show that a — 4 basic residue (i.e. 4 residues N-terminal to the site of cleavage) facilitates proinsulin conversion in the constitutive pathway, and that arginine is preferred over lysine.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00400813
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  • 3
    Electronic Resource
    Electronic Resource
    Proinsulin processing in the regulated and the constitutive secretory pathway (1994)
    Springer
    Diabetologia 37 (1994), S. S65 
    Details
    ISSN: 1432-0428
    Keywords: Proinsulin ; conversion ; conversion intermediates ; insulin ; beta cell ; secretory pathways
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Proinsulin is converted to insulin in betacell granules. Conversion involves endoproteolytic cleavage at the two pairs of basic residues linking the insulin A- and B-chains to C-peptide. The sequence of events leading to complete conversion differs from one proinsulin species to the next. In man, the structure of the proinsulin molecule is such as to favour cleavage at the B-chain/C-peptide junction leading to the generation of des-31,32 split proinsulin as the predominant, naturally occurring conversion intermediate. Under normal circumstances, proinsulin conversion is largely completed before secretion, and neither the intact prohormone nor conversion intermediates are thus encountered in large quantities in the circulation. In some pathological situations, including non-insulin-dependent diabetes, insulinoma and familial hyperproinsulinaemia, unusually high ratios of des-31,32 split proinsulin and/or proinsulin to insulin have been reported. As we understand the biochemistry of proinsulin conversion in increasingly fine molecular detail, it should become possible to make use of such unusual ratios to provide insight into lesions underlying altered beta-cell function in disease states.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00400828
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  • 4
    Electronic Resource
    Electronic Resource
    Trafficking of non-regulated secretory proteins in insulin secreting (INS-1) cells (2000)
    Springer
    Diabetologia 43 (2000), S. 1157-1164 
    Details
    ISSN: 1432-0428
    Keywords: Keywords GFP ; insulin ; regulated secretory pathway ; secretory granules ; secretory alkaline phosphatase.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Aims/hypothesis. Sorting of proinsulin to the regulated secretory pathway of pancreatic beta cells and retention of insulin in dense-core granules of this pathway is remarkably efficient. To monitor the specificity of these events, the secretion of two exogenous secretory proteins not known to carry information for sorting or retention in the regulated pathway was investigated in INS-1 cells. Methods. SEGFP, a fusion protein consisting of a signal peptide N-terminal to EGFP (mutant green fluorescent protein with enhanced fluorescence) and secreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transfection and by infection with recombinant adenovirus, respectively. Secretion of SEGFP was monitored by quantitative western blotting and that of SEAP by its activity. Results. Secreted alkaline phosphatase showed high basal secretion (6.6 % total) but only modest (3.6-fold) stimulation of secretion by secretagogues, in keeping with secretion largely through the constitutive pathway. By contrast SEGFP had a secretory pattern similar to insulin, with low basal secretion (0.8 % total) and 16-fold stimulation by secretagogues. Granular localization of SEGFP was confirmed by high resolution electron microscopy immunocytochemistry. Pulse-chase experiments indicated retention of SEGFP in granules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked oligomers. This could be implicated in its regulated secretion. Conclusion/interpretation. These data indicate that in INS-1 cells SEGFP, but not SEAP, is unexpectedly handled as a regulated secretory protein and stored along with insulin in granules. This raises questions about the specificity and mechanism of the sorting of proteins to granules in INS-1 cells or their retention therein or both. [Diabetologia (2000) 43: 1157–1164]
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001250051507
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  • 5
    Electronic Resource
    Electronic Resource
    Mobilization of subcutaneously injected tritiated insulin in rats: Effects of muscular exercise (1978)
    Springer
    Diabetologia 15 (1978), S. 133-140 
    Details
    ISSN: 1432-0428
    Keywords: Exercise ; tritiated insulin ; subcutaneous insulin injections ; exercise-induced hypoglycaemia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Previous studies in man and pancreatectomized dogs have indicated that alterations of the pharmacokinetics of subcutaneously injected insulin during physical activity may contribute to exercise-induced hypoglycaemia in insulin-treated diabetic patients. We have directly measured the appearance of subcutaneously injected insulin in the circulation and assessed its distribution to different tissues using a recently developed semisynthetic homogeneous [3H]insulin as a tracer. Following subcutaneous injection in rats of [3H]insulin in amounts insufficient to exert significant biological activity in intact animals, circulating levels of exogenous insulin were measured as plasma radioactivity co-migrating with insulin during gel filtration chromatography. Strenuous treadmill running accelerated the mobilization of subcutaneously injected [3H] insulin and resulted in a significant elevation of circulating levels of exogenous insulin early during exercise, followed by decreased levels in the post-exercise period. In addition, exercise induced a redistribution of 3H radioactivity in tissues, mainly increasing that found in skeletal muscle. This direct demonstration of altered pharmacokinetics of subcutaneously injected insulin during exercise provides, at least in part, a mechanism for the exercise-induced hypoglycemia seen following insulin injections in animals and during insulin treatment in man.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00422259
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  • 6
    Electronic Resource
    Electronic Resource
    High-performance liquid chromatography (HPLC): A rapid, flexible and sensitive method for separating islet proinsulin and insulin (1986)
    Springer
    Diabetologia 29 (1986), S. 893-896 
    Details
    ISSN: 1432-0428
    Keywords: HPLC ; proinsulin processing ; insulin biosynthesis ; isolated islets ; islet function
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Evaluating islet function in vitro involves studying both insulin biosynthesis and release. For the former, it is necessary to resolve insulin from its precursor, proinsulin. This has been achieved in the past by various procedures, each of which suffers from major drawbacks in terms of resolution and the time involved. We show here that reversed phase high-performance liquid chromatography (HPLC) outperforms previous methods for separating proinsulin from insulin in is let extracts without any prepurification or concentration steps. This HPLC method is rapid (90 min for a complete cycle, including washing the column) and reproducible, while allowing for unambiguous separation and quantification of proinsulin and insulin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00870146
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  • 7
    Electronic Resource
    Electronic Resource
    Resistance of the insulin crystal to lysosomal proteases: implications for pancreatic B-cell crinophagy (1987)
    Springer
    Diabetologia 30 (1987), S. 348-353 
    Details
    ISSN: 1432-0428
    Keywords: Insulin ; proinsulin ; crinophagy ; insulin crystals ; degradation ; lysosomes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Insulin is thought to be chemically stabilized within β-granules in the crystal form. The other major products of the β-granule, proinsulin and C-peptide, by contrast, are not thought able to crystallize. The physico-chemical properties of peptides in soluble or crystalline form are dramatically different. The ability of insulin to crystallize in the β-granule might thus explain why this peptide, but not proinsulin/Cpeptide, remains stable even after its introduction into lysosomes as occurs during granulolysis (crinophagy). We have now studied this by exposing proinsulin or insulin to lysosomal proteases in vitro. 125I-insulin in soluble form was found to be degraded at the same rate as 125I-proinsulin. Strikingly, however, when the labelled insulin was crystallized, its rate of degradation was decreased from 1.9 to 0.2 pmol/min. We take these data as confirmation that the insulin crystal is resistant to degradation, thereby possibly accounting for (a) the presence of insulin immunoreactivity within multigranular bodies, and (b) the unusually slow rate of degradation of insulin within B cells compared with that of other hormones in their cells of origin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00299029
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  • 8
    Electronic Resource
    Electronic Resource
    Activation of liver and muscle insulin receptor tyrosine kinase activity during in vivo insulin administration in rats (1990)
    Springer
    Diabetologia 33 (1990), S. 77-83 
    Details
    ISSN: 1432-0428
    Keywords: Hyperinsulinaemic glucose clamp ; skeletal muscle ; liver ; insulin receptors ; tyrosine kinase ; insulin resistance ; β-subunit C-terminus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We have studied autophosphorylation and tyrosine kinase activity of the insulin receptor purified from liver and muscle of fasted rats before and after infusion of insulin (100 mU/h) during a 2.5 h glucose clamp. Recovery of insulin receptors and insulin binding to the solubilised receptors was unaffected by the glucose clamp. Autophosphorylation of the insulin receptor β subunit was increased in liver receptors prepared from rats at the end of the glucose clamp compared to rats in the basal state both in the absence of insulin in vitro (109% increase, p〈0.001) and after in vitro stimulation with 10−7 mol/l insulin (clamped vs fasted; 96% increase, p〈0.001). Insulin (10−7 mol/l) stimulated autophosphorylation was also increased in muscle receptor preparations from clamped rats compared with rats in the basal state (58% increase, p〈0.05). In both liver and muscle receptors, the clamp increased the amount of [32P]-phosphate incorporated into the β subunit without changing the sensitivity of the insulin stimulation. HPLC analysis of the tryptic phosphopeptides derived from the β subunit after insulin stimulated autophosphorylation of liver receptors revealed an increase of 32P in all phosphorylation sites without any change in the overall pattern. Tyrosine kinase activity of liver and muscle insulin receptors from clamped rats was also increased approximately twofold (p〈0.05) when analysed using a synthetic substrate (poly Glu4 Tyr1). Our results support the notion that the insulin receptor exists in an active and inactive form, and that elevated plasma insulin concentrations increases the proportion of active receptors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00401044
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  • 9
    Electronic Resource
    Electronic Resource
    High-performance liquid chromatography analysis of circulating insulins distinguishes between endogenous insulin production (a potential pitfall with streptozotocin diabetic rats) and islet xenograft function (1990)
    Springer
    Diabetologia 33 (1990), S. 457-461 
    Details
    ISSN: 1432-0428
    Keywords: Microencapsulated ; porcine ; islets ; streptozotocin ; diabetic rats ; pancreas regeneration ; bioartificial pancreas ; high performance liquid chromatography
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Porcine islets of Langerhans were microencapsulated according to the alginate-polylysine procedure, and implanted into the peritoneal cavity of 15 streptozotocin-induced (70 mg/kg) diabetic rats (6000 microencapsulated islets per rat). In four animals, a sustained decrease in plasma glucose level below 8.3 mmol/l was observed for up to nine months. However, it was possible to recover microcapsules from the peritoneal cavity of only one rat, and they were found to be damaged and containing no detectable tissue. When insulin in the plasma of three of these animals was analysed by reversed phase high-performance liquid chromatography, only rat insulins I and II, but not porcine insulin was detectable, indicating unambiguously that at the time of analysis, the correction of diabetes in these animals was due to the function of the recipient's own pancreas rather than the continued, long-term, function of the implanted porcine islets. These data confirm that in this model of diabetes, function of the host pancreas can resume following islet transplantation, leading in turn to the potential for a major bias in the interpretation of the data. In the case of an islet xenograft, when the donor's and recipient's insulins can be separated by highperformance liquid chromatography, this non-invasive analytical method should prove useful for identifying the source of insulin in the circulation, and thus the relative functional status of the endogenous and transplanted islets.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00405105
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  • 10
    Electronic Resource
    Electronic Resource
    Structural domains and molecular lifestyles of insulin and its precursors in the pancreatic Beta cell (1991)
    Springer
    Diabetologia 34 (1991), S. 767-778 
    Details
    ISSN: 1432-0428
    Keywords: Preproinsulin ; proinsulin ; insulin ; C-peptide ; biosynthesis ; precursor processing ; protein trafficking ; protein degradation ; pancreatic Beta cell
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Insulin is both produced and degraded within the pancreatic Beta cell. Production involves the synthesis of the initial insulin precursor preproinsulin, which is converted to proinsulin shortly after (or during) translocation into the lumen of the rough endoplasmic reticulum. Proinsulin is then transported to the trans-cisternae of the Golgi complex where it is directed towards nascent secretory granules. Conversion of proinsulin to insulin and C-peptide arises within secretory granules, and is dependent upon their acidification. Granule contents are discharged by exocytosis in response to an appropriate stimulus. This represents the regulated secretory pathway to which more than 99% of proinsulin is directed in Beta cells of a healthy individual. An alternative route also exists in the Beta cell, the constitutive secretory pathway. It involves the rapid transfer of products from the Golgi complex to the plasma membrane for immediate release, with, it is supposed, little occasion for prohormone conversion. Even if delivered appropriately to secretory granules, not all insulin is released; some is degraded by fusion of granules with lysosomes (crinophagy). Each event in the molecular lifestyles of insulin and its precursors in the Beta cell will be seen to be governed by their own discrete functional domains. The identification and characterisation of these protein domains will help elucidate the steps responsible for delivery of proinsulin to secretory granules and conversion to insulin. Understanding the molecular mechanism of these steps may, in turn, help to explain defective insulin production in certain disease states including diabetes mellitus.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00408349
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