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Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells

Author:
Amao, Michiko, Kitahara, Yoshiro, Tokunaga, Ayaka, Shimbo, Kazutaka, Eto, Yuzuru, Yamada, Naoyuki
Source:
Analytical biochemistry 2015 v.472 pp. 45-51
ISSN:
0003-2697
Subject:
cultured cells, glucagon-like peptide 1, glucose, hormone secretion, liquid chromatography, monitoring, noninsulin-dependent diabetes mellitus, patients, secretin, sucralose, tandem mass spectrometry
Abstract:
Glucagon-like peptide-1 (GLP-1) is an incretin peptide that regulates islet hormone secretion. During recent years, incretin-based therapies have been widely used for patients with type 2 diabetes. GLP-1 peptides undergo N- and C-terminal processing for gain or loss of functions. We developed a method to quantify picomolar quantities of intact GLP-1 peptides using liquid chromatography–tandem mass spectrometry (LC–MS/MS). By employing this label-free selected reaction monitoring (SRM) method, we were able to analyze secreted GLP-11–37, GLP-17–37, and GLP-17–36amide from human enteroendocrine NCI-H716 cells after stimulation with nateglinide, glucose, and sucralose. The absolute total concentrations of secreted GLP-1 peptides at baseline and after stimulation with nateglinide, glucose, and sucralose were 167.3, 498.9, 238.3, and 143.1pM, respectively. Meanwhile, the ratios of GLP-11–37, GLP-17–37, and GLP-17–36amide to total GLP-1 peptides were similar (6±3, 26±3, and 78±5%, respectively). The SRM assay can analyze the concentrations of individual GLP-1 peptides and, therefore, is a tool to investigate the physiological roles of GLP-1 peptides. Furthermore, the molecular species secreted from NCI-H716 cells were unknown. Therefore, we performed a secretopeptidome analysis of supernatants collected from cultured NCI-H716 cells. Together with GLP-1 peptides, we detected neuroendocrine convertase 1, which regulates peptide hormones released from intestinal endocrine L-cells.
Agid:
6021339