Molecular Forms of Glucagon-Like Peptide-1 in Human Pancreas and Glucagonomas

Abstract

The structure of human preproglucagon, as deduced from nucleotide sequencing of the preproglucagon gene, contains two glucagon-like peptides (GLP-1 and GLP-2) in the portion C-terminal to glucagon. A rabbit antiserum was raised against synthetic GLP-1-(1-19) which had 20% cross-reactivity with synthetic GLP-1 and des-Gly37-GLP-1 amide, two possible forms of the GLP-1 whole molecule, but no significant cross-reactivity with glucagon or other pancreatic peptides. Immunocytochemistry revealed that the distribution of GLP-1-(1-19) immunoreactivity followed that of glucagon-like immunoreactivity in the normal human pancreas and in two human glucagon-secreting pancreatic tumors. Chromatography of human pancreas extracts on Sephadex G-50 gave peaks of cross-reactivity at Kav values of 0.06-0.16, 0.34-0.39, 0.54-0.58 (the elution position of synthetic GLP-1), and 0.64-0.70. The concentration of immunoreactivity in the Kav 0.54-0.58 peak measured by RIA using GLP-1 or des-Gly37-GLP-1 amide as standard was 94 +/- 7 pmol/g (mean +/- SEM), while the total pancreatic glucagon content was 4.8 +/- 0.8 nmol/g. One extract of a human glucagon-secreting pancreatic tumor contained a prominent peak of GLP-1-(1-19) peptide cross-reactivity with properties identical to those of GLP-1 or des-Gly37-GLP-1 amide on gel filtration and reverse phase high pressure liquid chromatography, but another tumor contained a preponderance of cross-reactive forms of greater molecular size. Pretreatment plasma from three patients with radiological and biochemical evidence of glucagon-secreting tumors contained a peak of cross-reactivity with the chromatographic properties of intact GLP-1. The low concentrations of intact GLP-1 in normal pancreas compared with pancreatic glucagon concentrations suggest that the majority of the proglucagon is cleaved in a manner that does not produce GLP-1, as defined by its delimiting pairs of basic amino acid residues.

A local glucagon-like peptide 1 (GLP-1) system in human pancreatic islets

“…GLP-1-like immunoreactivity was detected by a polyclonal and a monoclonal anti-GLP-1 antibody reacting, respectively, with the mid to C terminal region of GLP-1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] and the amidated C-terminus of the peptide (see the Methods). These antibodies generated similar staining patterns with NCI-H716 cells and human islets (ESM Figs 2 and 3).…”

“…The corresponding spectra of the GLP-1(7-37) and GLP-1 (7-36) tryptic sequences were matched with the putative digested sequences of the peptides using the sequence editor implemented on BIOTOOLS. GLP-1(7-37) and GLP-1(7-36) peptides were identified by the species at m/z 1105 (aa [21][22][23][24][25][26][27][28] and at m/z 2098 (aa [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20].…”

“…Primary antibodies were: guinea pig polyclonal anti-swine insulin (A0564; DAKO, Carpinteria, CA, USA); mouse monoclonal anti-human/ mouse glucagon (MAB1249, clone 181402; R&D Systems, Abingdon, UK); rat monoclonal anti-somatostatin (ab30788; Abcam, Cambridge, UK); rabbit polyclonal anti-GLP-1 (ab22625; Abcam), which reacts with the mid to C terminal region of GLP-1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], enabling immunoreactivity with N terminal truncated and C terminally extended forms of GLP-1; mouse monoclonal anti-GLP-1 (HYB 147-06; BioPorto Diagnostic, Gentofte, Denmark), which is specific for the amidated C-terminus of the peptide [20]; and rabbit polyclonal antibody to PC1/3 and PC2 (Chemicon, Rosemont, IL, USA). Secondary antibodies were: Alexa Fluor 488: goat anti-guinea pig IgG, goat anti-rabbit IgG, and goat anti-mouse IgG; and Alexa Fluor 594: goat anti-mouse IgG and goat antirabbit IgG (all from Invitrogen, Molecular Probes, Leiden, the Netherlands).…”

“…In the mid-1980s, investigators using antibodies against GLP-1 and electron microscopic immunocytochemistry observed molecular forms of GLP-1 in the human pancreas [9,10]. Later, fully processed GLP-1 was visualised in rat pancreatic extracts by chromatographic analysis and radioimmunoassays [11].…”

Glucagon-like peptide-1 but not glucagon-like peptide-2 stimulates insulin release from isolated rat pancreatic islets

“…This substantial conservation in evolution and the close sequence homology to the glucagon molecule (GLP-I: 48% identical residues; GLP-2: 38%) indicates that GLP-1 and GLP-2 may play an important role of their own as hormones or local modulators. Recently they have been detected by specific radioimmunoassays in glucagonomas and in rat pancreatic islets [6], although they have not to date been isolated at the peptide level. Very little is known about a specific biological activity of these peptides.…”