Oxyntomodulin Identified as a Marker of Type 2 Diabetes and Gastric Bypass Surgery by Mass-spectrometry Based Profiling of Human Plasma

Abstract

Low-abundance regulatory peptides, including metabolically important gut hormones, have shown promising therapeutic potential. Here, we present a streamlined mass spectrometry-based platform for identifying and characterizing low-abundance regulatory peptides in humans. We demonstrate the clinical applicability of this platform by studying a hitherto neglected glucose- and appetite-regulating gut hormone, namely, oxyntomodulin. Our results show that the secretion of oxyntomodulin in patients with type 2 diabetes is significantly impaired, and that its level is increased by more than 10-fold after gastric bypass surgery. Furthermore, we report that oxyntomodulin is co-distributed and co-secreted with the insulin-stimulating and appetite-regulating gut hormone glucagon-like peptide-1 (GLP-1), is inactivated by the same protease (dipeptidyl peptidase-4) as GLP-1 and acts through its receptor. Thus, oxyntomodulin may participate with GLP-1 in the regulation of glucose metabolism and appetite in humans. In conclusion, this mass spectrometry-based platform is a powerful resource for identifying and characterizing metabolically active low-abundance peptides.

Keywords: GLP-1; Gut hormones; Low-abundant peptides; Mass-spectrometry; Proteomics.

Fig. 1

A streamlined platform for detection of low-abundant peptides. This figure summarizes the key components in our mass-spectrometry based platform. Human blood or tissue samples are purified using C18 materials and subsequently proteins are separated using high-performance liquid chromatography. Different proteases are then added to the 96 wells to initiate protein digestion and formation of peptides. The samples are subsequently purified using C18 materials and subjected to liquid chromatography coupled to mass spectrometry (LC–MS/MS) followed by data analysis using MaxQuant.

Fig. 2

Proteomic profiling of plasma from gastric bypass operated patients and human gut biopsies. A: Illustrates intensities of individual peptides identified of oxyntomodulin and glicentin in human gut, plasma and from recombinant controls. B: Illustrates sequence coverage of oxyntomodulin and glicentin in human gut, plasma and recombinant controls. Evidence for coverage of the respective hormone is depicted as the summed peptide intensities per amino acid for all employed proteases (see Material & Methods). 10 biological replicates and 3 technical replicates are shown.

Fig. 3

Oxyntomodulin responses are blunted in type 2 diabetic subjects and 10-fold elevated after gastric bypass surgery. This figure illustrates the concentrations of oxyntomodulin (red) and glicentin (black) in A: 10 healthy subjects (squares, full line) and 10 patients with type 2 diabetes (triangles, dotted line) during a standard OGTT challenge; B: Illustrates calculated incremental AUC of data presented in A; 18 obese non diabetic patients during a standardized meal challenge before (C), 1 month after gastric bypass (D) and 6 months after gastric bypass (E). F: _tAUC calculated based on data from C,D,E. Data are mean ± SEM. Asterisk (*) represents statistical significant differences by a unpaired t-test (B) or one-way ANOVA correcting for multiple testing by Bonferroni post hoc analysis (F).

Fig. 4

Oxyntomodulin is co-distributed, co-secreted with GLP-1 and act through same receptor. A: Concentrations of extractable oxyntomodulin (white boxes) and glicentin (black boxes) normalized to protein content in GLUtag, NCI-H716 and STC-1 cells, the most frequently used cell models in incretin biology. B: Total GLP-1 tissue concentrations (black boxes) and oxyntomodulin concentrations (red boxes) increase significantly (P ˂ 0.001) along the gastrointestinal tract in mice (n = 10). C: Secretion of GLP-1 (black curve) and oxyntomodulin (red curve) from perfused proximal small intestine (n = 4). Secretion was significantly (P ˂ 0.05) increased by infusion of neuromedin C (10 mM) and KCL (70 mM). D: Averaged levels of GLP-1 and oxyntomodulin during basal period compared to stimulation-period with either neuromedin C (grey) or KCl (black). Oxyntomodulin and GLP-1 secretion increased in parallel during both neuromedin C and KCl stimulation. E: Correlation plot using data from C; the correlation coefficient was 0.91 (R²). Data are illustrated as mean ± SEM. * represent statistical significant differences using a one-way ANOVA correcting for multiple testing by Bonferroni post hoc analysis (B) or a paired t-test (C). Differences between GLP-1 and oxyntomodulin were not significant. F: Levels of oxyntomodulin in buffer ± DPP-4 inhibition and human plasma ± DPP-4 inhibition. Measured levels of oxyntomodulin were higher (P = 0.011) in plasma but not in buffer (P = 0.34) upon DPP-4 inhibition.