Measurement of Gastrointestinal Hormones.

Towards the end of the 20th century, the number of subjects with diabetes and obesity rose exponentially. The discoveries of insulin- and appetite-modulating chemical signals, including glucagon-like peptide-1 (GLP-1), secreted from the gastrointestinal system, led to development of a new group of drugs which now are being used for glucose-lowering therapy and weight loss. Understanding of the physiology of gut derived signals and their pathophysiologi-cal importance requires accurate measurements of their circulat-ing levels. However, the assessment of these gut-derived hor-mones has been hampered by numerous preanalytical and analyti-cal challenges. We focused on three members of the proglucagon family; glucagon, oxyntomodulin and GLP-1, aiming to meet both preanalytical and analytical challenges and to elucidate their implication in diseases including diabetes. First, we studied (Study 1) the preanalytical and storage conditions of GLP-1 and glucagon in humans, demonstrating that inappropriate sample handling may cause up to 50% variation in the RESULTS. Using robust meas-uring METHODS ensuring optimal conditions for preanalytical han-dling of these peptides, we then focused on plasma concentra-tions of glucagon and oxyntomodulin in different clinical condi-tions, including type 2 diabetes and bariatric surgery, because abnormal secretion of these hormones may represent early and specific signs of altered glucose metabolism. To that end, we developed an unbiased mass-spectrometry based platform for detection of low-abundant peptides, including the gut hormones (Study 2). Using the platform, we validated a new method for the measurement of oxyntomodulin, and in a series of in vitro, ex vivo, and clinical studies, we demonstrated that oxyntomodulin is co-distributed and co-secreted in response to glucose with GLP-1 and is degraded by dipeptidyl peptidase 4. Because oxyntomodulin has both GLP-1-like and glucagon-like bioactivity, the secretion of this hormone is of interest in both type 2 diabetes and bariatric sur-gery. Furthermore, using these newly developed METHODS, we subsequently were able to establish that elevated plasma concen-trations of glucagon (hyperglucagonemia) in diseases (Study 3) may be due to either a) increased secretion of fully processed glucagon, as in subjects with diabetes or b) secretion of N-terminally elongated molecular forms (Study 4) in conditions including bariatric surgery and in diseases affecting the kidneys. This glucagon variant may be of importance for glucose homeo-stasis, as we were able to show that it, unexpectedly, activates the glucagon receptor, leading to increased glycogenolysis in hepatocytes and insulin secretion from pancreatic beta-cells. In summary, accurate measurements of gut-derived hormones are indeed crucial for understanding their biology in health and as well in disease. Mass-spectrometry based plasma proteomics is a powerful tool for the validation of these METHODS.