Oxidation of rat insulin II, but not I, leads to anomalous elution profiles upon HPLC analysis of insulin‐related peptides

Gross, David J.; Skvorak, Anne; Hendrick, G. K.; Weir, Gordon C.; Villa‐Komaroff, Lydia; Halban, Philippe A.

doi:10.1016/0014-5793(88)81062-7

Cited by 19 publications

(19 citation statements)

References 16 publications

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“…The comparison of the amount of insulin eluting from :y ofextraction HPLC with that injected confirmed our previous observations ofquan--insulin mixed titative recovery using similar HPLC systems ( 16,19,29,30 Relative amounts ofmouse insulins I and II and ofhuman insulin in thepancreas ofcontrol and transgenic mice. Previous characterization of the transgenic mice indicated that human insulin mRNA was present in islets and that human C-peptide (presumably derived from correctly processed human proinsulin) was secreted into the circulation (20).…”

Section: Biochemistrysupporting

confidence: 85%

“…Only two immunoreactive peaks were seen for control pancreas (top), eluting as mouse insulins I and II, with a third, minor peak eluting earlier than the two insulins. This material has been identified as mouse insulin II oxidized at methionine B29 (29), and for the purposes of calculating the relative amounts of the various insulin species is considered as mouse insulin II. As expected for the pancreas of a mouse ( 15,19), there was more than twice as much insulin II than I.…”

Section: Biochemistrymentioning

confidence: 99%

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Adaptation to supraphysiologic levels of insulin gene expression in transgenic mice: evidence for the importance of posttranscriptional regulation.

Schnetzler

Murakawa²,

Abalos³

et al. 1993

J. Clin. Invest.

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Insulin production was studied in transgenic mice expressing the human insulin gene under the control of its own promoter. Glucose homeostasis during a 48-h fast was similar in control and transgenic mice, with comparable levels of serum immunoreactive insulin. Northern blot and primer extension analyses indicated that more than twice as much insulin mRNA is present in pancreata from transgenic mice. Primer extension analysis using oligonucleotides specific for mouse insulins I and II or for human insulin, showed that the excess insulin mRNA was due solely to expression of the foreign, human insulin gene. The ratio of mRNA for mouse insulin I and II was unaffected by coexpression of human insulin. There were coordinate changes in the levels of all three mRNA during the 48-h fast, or after a 24-h fast followed by 24-h refeed. Despite the supraphysiologic levels of insulin mRNA in the transgenic mice, their pancreatic content of immunoreactive insulin was not significantly different from controls. The comparison of the relative levels of human and mouse insulin mRNAs with their peptide counterparts (separated by HPLC) indicates that the efficiency of insulin production from mouse insulin mRNA is greater than that from human, stressing the importance of posttranscriptional regulatory events in the overall maintenance of pancreatic insulin content. (J. Clin. Invest. 1993. 92:272-280.)

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Section: Biochemistrysupporting

confidence: 85%

Section: Biochemistrymentioning

confidence: 99%

Adaptation to supraphysiologic levels of insulin gene expression in transgenic mice: evidence for the importance of posttranscriptional regulation.

Schnetzler

Murakawa²,

Abalos³

et al. 1993

J. Clin. Invest.

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“…In order to separate radiolabeled proinsulin from insulin, aiiquots of islet extracts or of incubation buffer were analyzed by reversed phase HPLC as described previously (Haiban et ai., 1986;Gross et al, 1988;Sizouenko and Haihan, 1991). In brief, samples were injected onto a Beclmum Ultrasphere ODS 5/~m column (dimensions 4.5 x 250 ram) connected to a Beckman System Gold HPLC apparatus, using TEAP (20 mM triethylamine, 50 mM phosphoric acid, 50 mM perchloric acid, pH 3.0) as buffer A and 90% acetum'trile/10% H20 as buffer B.…”

Section: Methodsmentioning

confidence: 99%

pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle.

Orci

Halban

Perrelet

et al. 1994

The Journal of Cell Biology

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Abstract. By quantitative immunoelectron microscopy and HPLC, we have studied the effect of disrupting pH gradients, by ammonium chloride, on proinsulin conversion in the insulin-producing B-cells of the islets of Langerhans. Proinsulin content and pH in single secretory vesicles were measured on consecutive serial sections immunostained alternately with antiproinsulin or anti-dinitrophenol (to reveal the pHsensitive probe DAMP) antibodies. Radioactively labeled proinsulin, proinsulin cleavage intermediates, and insulin were quantitated by HPLC analysis of extracts of islets treated in the same conditions. Cleavage at the C-peptide/A-chain junction is significantly less sensitive to pH gradient disruption than that of the B-chain/C-peptide junction, but the range of pH and proinsulin content in individual vesicles indicate that both cleavages occur in the same vesicle released from the TGN.IDIFICATIOr~ of many of the intracellular membrane bounded compartments that are created on the endocytotic and secretory pathways is coupled to orderly protein processing and membrane traffic. For example, during receptor-mediated endocytosis, low pH promotes the dissociation of the ligand from its receptor before fusion of the endosome with the lysosome. Acidification of vesicles originating from the TGN activates proteolytic enzymes that generate functional hormones by cleaving precursor proteins (e.g., prohormones) as they emerge from the Golgi (for review see Anderson and Orci, 1986;Mellman et al., 1986).Acidification is mediated by a membrane (H+)-ATPase that is able to maintain variable concentrations of protons within the vesicle lumen. As a consequence, the pH of sequential compartments in an endocytic or secretory pathway can change from 7.0 to as low as 4.5 (for review see AIAwqati, 1986). There is also clear evidence that in certain cells the pH of a compartment is tightly regulated (Mallya et al., 1992). Therefore, pH is often used to modulate chemical reactions that take place within intracellular vesicles.In the insulin-producing B-cell of the islet of Langerhans, biochemical and immunocytochemical studies have shown that the synthesis of proinsulin in the endoplasmic reticulum and its transport to the TGN occur in a relatively neutral pH environment. The conversion of proinsulin to insulin, however, is initiated in acidic (~pH 6.0) clathrin-coated secre-

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“…Unlabeled samples eluted from SepPak cartridges or radiolabeled products after immunoprecipitation were further analyzed by reversed-phase HPLC. The system used an Altex Ultrasphere ODS 5-,um column and an acetonitrile/TEAP buffer system as described (16)(17)(18). For radioactive samples, 1-ml fractions were collected and added to 10 ml of liquid scintillation cocktail (Biocount from Research Products International) and the radioactivity was then measured in a liquid scintillation counter.…”

Section: Methodsmentioning

confidence: 99%

“…Further investigation of this family revealed a single nucleotide transversion in the codon for residue 10 of the proinsulin B-chain (CAC to GAC), predicting a substitution of aspartic acid for histidine at this position, which is distant from the dibasic cleavage sites (5). To understand why this mutation leads to hyperproinsulinemia, we introduced an identical mutation into the rat insulin II gene and utilized the ability of AtT-20 cells transfected with the insulin gene to correctly process and target the native insulin gene product, leading to regulated secretion of mature insulin (6)(7)(8) Mutagenesis and Construction of Plasmids. The EcoRI/ BamHI fragments from the plasmid pRI2-SV-40-46 (9), were cloned into EcoRI/BamHI-digested M13mpl9 replicative form and used to transform JM109 cells.…”

mentioning

confidence: 99%

Partial diversion of a mutant proinsulin (B10 aspartic acid) from the regulated to the constitutive secretory pathway in transfected AtT-20 cells.

Gross

Halban²,

Kahn³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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A patient with type II diabetes associated with hyperproinsulinemia has been shown to have a point mutation in one insulin gene allele, resulting in replacement of histidine with aspartic acid at position 10 of the B-chain. To investigate the basis of the proinsulin processing defect, we introduced an identical mutation in the rat insulin II gene and expressed both the normal and the mutant genes in the AtT-20 pituitary corticotroph cell line. Cells expressing the mutant gene showed increased secretion of proinsulin relative to insulin and rapid release of newly synthesized proinsulin. Moreover, the mutant cell lines did not store the prohormone nor did they release it upon stimulation with secretagogues. These data indicate that a significant fraction of the mutant prohormone is released via the constitutive secretory pathway rather than the regulated pathway, thereby bypassing granule-related processing and regulated release.Familial hyperproinsulinemia is a genetic disorder characterized by increased serum levels of proinsulin-like material relative to insulin in affected individuals. In two kindreds, an amino acid substitution for arginine at the dibasic cleavage site joining the C-peptide to the A-chain leads to defective processing and increased release of partially cleaved proinsulin (1-3). In contrast, a third family with hyperproinsulinemia has been described in which proinsulin is readily cleaved in vitro to mature insulin and C-peptide by tryptic digestion (4). Further investigation of this family revealed a single nucleotide transversion in the codon for residue 10 of the proinsulin B-chain (CAC to GAC), predicting a substitution of aspartic acid for histidine at this position, which is distant from the dibasic cleavage sites (5). To understand why this mutation leads to hyperproinsulinemia, we introduced an identical mutation into the rat insulin II gene and utilized the ability of AtT-20 cells transfected with the insulin gene to correctly process and target the native insulin gene product, leading to regulated secretion of mature insulin (6)(7)(8) Mutagenesis and Construction of Plasmids. The EcoRI/ BamHI fragments from the plasmid pRI2-SV-40-46 (9), were cloned into EcoRI/BamHI-digested M13mpl9 replicative form and used to transform JM109 cells. Recombinant DNA with 193-nucleotide insert encoding the B-chain, the first 6 amino acids of the C-peptide, and part of the second intron was isolated and sequenced by the dideoxynucleotide chaintermination method (10). After correct sequence and orientation were verified, site-directed mutagenesis using a 14-mer (5'-ACCAACTCAGAACC-3') was performed with the Amersham oligonucleotide-directed system according to the manufacturers' recommendations. Mutants were identified by sequencing; the entire fragment was sequenced to ensure that no other mutations were introduced during the procedure. The mutant 193-base-pair (bp) EcoRI/BamHI fragment, the 1058-bp EcoRI/BamHI fiagment from pRI2-SV40-46, and BamHIdigested, phosphatase-treated pRI2-SV40-46-Del (t...

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Oxidation of rat insulin II, but not I, leads to anomalous elution profiles upon HPLC analysis of insulin‐related peptides

Cited by 19 publications

References 16 publications

Adaptation to supraphysiologic levels of insulin gene expression in transgenic mice: evidence for the importance of posttranscriptional regulation.

Adaptation to supraphysiologic levels of insulin gene expression in transgenic mice: evidence for the importance of posttranscriptional regulation.

pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle.

Partial diversion of a mutant proinsulin (B10 aspartic acid) from the regulated to the constitutive secretory pathway in transfected AtT-20 cells.

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