Postprandial Dynamics of Proglucagon Cleavage Products and Their Relation to Metabolic Health

Abstract

Introduction: While oral glucose ingestion typically leads to a decrease in circulating glucagon levels, a substantial number of persons display stable or rising glucagon concentrations when assessed by radioimmunoassay (RIA). However, these assays show cross-reactivity to other proglucagon cleavage products. Recently, more specific assays became available, therefore we systematically assessed glucagon and other proglucagon cleavage products and their relation to metabolic health.

Research design and methods: We used samples from 52 oral glucose tolerance tests (OGTT) that were randomly selected from persons with different categories of glucose tolerance in an extensively phenotyped study cohort.

Results: Glucagon concentrations quantified with RIA were non-suppressed at 2 hours of the OGTT in 36% of the samples. Non-suppressors showed lower fasting glucagon levels compared to suppressors (p=0.011). Similar to RIA measurements, ELISA-derived fasting glucagon was lower in non-suppressors (p<0.001). Glucagon 1-61 as well as glicentin and GLP-1 kinetics were significantly different between suppressors and non-suppressors (p=0.004, p=0.002, p=0.008 respectively) with higher concentrations of all three hormones in non-suppressors. Levels of insulin, C-peptide, and free fatty acids were comparable between groups. Non-suppressors were leaner and had lower plasma glucose concentrations (p=0.03 and p=0.047, respectively). Despite comparable liver fat content and insulin sensitivity (p≥0.3), they had lower 2-hour post-challenge glucose (p=0.01).

Conclusions: Glucagon 1-61, glicentin and GLP-1 partially account for RIA-derived glucagon measurements due to cross-reactivity of the assay. However, this contribution is small, since the investigated proglucagon cleavage products contribute less than 10% to the variation in RIA measured glucagon. Altered glucagon levels and higher post-challenge incretins are associated with a healthier metabolic phenotype.

Trial registration: ClinicalTrials.gov NCT01947595.

Keywords: Glucagen-like peptides; glicentin; glucagon; insulin; metabolism.

Conflict of interest statement

Outside of the current work, RW does report lecture fees from Novo Nordisk, Sanofi-Aventis and travel grants from Eli Lilly. He served on the advisory board for Akcea Therapeutics, Daiichi Sankyo, Sanofi-Aventis and NovoNordisk. Outside of the current work, AF reports lecture fees and advisory board membership from Sanofi, Novo Nordisk, Eli Lilly, and AstraZeneca. In addition to his current work, AB reports lecture fees from AstraZeneca, Boehringer Ingelheim, and NovoNordisk. He served on advisory boards of AstraZeneca, Boehringer Ingelheim and NovoNordisk. Outside of the current work, MH, reports research grants from Boehringer Ingelheim and Sanofi (both to the University Hospital of Tübingen), advisory board for Boehringer Ingelheim, and lecture fees from Amryt, Novo Nordisk and Boehringer Ingelheim. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Wagner, Eckstein, Fritsche, Prystupa, Hörber, Häring, Birkenfeld, Peter, Fritsche and Heni.

Figures

Figure 1

Schematic presentation of proglucagon and proglucagon cleavage products. Numbers indicate amino acid positions of cleavage sites. Antibodies schematically indicate epitopes that are used by the commercial immunoassays applied in our measurements (as provided by the manufacturer).

Figure 2

Concentrations of investigated analytes in the groups of glucagon suppressors and non-suppressors during the OGTT. The respective analyte is indicated in the box. (A: glucagon measured by radioimmunoassay, B: glucagon measured by ELISA, C: glucagon 1-61, D: glicentin, E: GLP-1, F: glucose, G: C-peptide, H: insulin, I: free fatty acids). Lines represent means with standard errors. Circles indicate data points for suppressors, triangles for non-suppressors, p-values were calculated with linear mixed models. N=52.

Figure 3

Bland-Altman plot of the RIA- and ELISA-measured glucagon. Differences in glucagon measurements between the two assays are plotted against mean glucagon values. The dashed lines represent the mean, the solid lines depict the lines of agreement calculated as mean ± 1.96 times the SD of this difference. N=156 measurements from 52 oGTTs.

Figure 4

Estimated model-based relative contribution of proglucagon cleavage products to the variance of RIA-derived glucagon measurements. We used linear mixed models with the participant as a random intercept and the OGTT-time point as fixed effects. Marginal R squared was calculated to describe the proportion of variance in the outcome variable explained by the fixed effect. Removing each factor separately, we determined the percentage of its respective contribution to RIA-derived glucagon measurements that are presented here as a bar chart.