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Plasma concentrations of glucagon, GLP-1 and GIP are reported in numerous clinical trials as outcome measures but preanalytical guidelines are lacking. We addressed the impact of commonly used blood containers in metabolic research on measurements of glucagon, GLP-1 and GIP in humans. Seventeen overweight individuals were subjected to an overnight fast followed by an intravenous infusion of amino acids to stimulate hormonal secretion. Blood was sampled into five containers: EDTA-coated tubes supplemented with DMSO (control), a neprilysin inhibitor, aprotinin (a kallikrein inhibitor) or a DPP-4 inhibitor, and P800 tubes. Plasma was kept on ice before and after centrifugation and stored at −80 Celsius until batch analysis using validated sandwich ELISAs or radioimmunoassays (RIA). Measures of fasting plasma glucagon did not depend on sampling containers, whether measured by ELISA or RIA. Amino acid-induced hyperglucagonemia was numerically higher when blood was collected into P800 tubes or tubes with aprotinin. The use of p800 tubes resulted in higher concentrations of GLP-1 by RIA compared to control tubes but not for measurements with sandwich ELISA. Plasma concentrations of GIP measured by ELISA were higher in control tubes and negatively affected by P800 and the addition of aprotinin. The choice of blood containers impacts on measurements of plasma concentrations of glucagon, GLP-1 and GIP, and based on this study, we recommend using EDTA-coated tubes without protease inhibitors or P800 tubes for measurements of glucagon, GLP-1 and GIP in clinical trials. [ABSTRACT FROM AUTHOR]

Summary Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in β cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from the gastrointestinal tract. It is best known for its glucose-dependent insulinotropic effects. GLP-1 is secreted in its intact (active) form (7-36NH2) but is rapidly degraded by the dipeptidyl peptidase 4 (DPP-4) enzyme, converting >90% to the primary metabolite (9-36NH2) before reaching the targets via the circulation. Although originally thought to be inactive or antagonistic, GLP-1 9-36NH2 may have independent actions, and it is therefore relevant to be able to measure it. Because reliable assays were not available, we developed a sandwich ELISA recognizing both GLP-1 9-36NH2 and nonamidated GLP-1 9-37. The ELISA was validated using analytical assay validation guidelines and by comparing it to a subtraction-based method, hitherto employed for estimation of GLP-1 9-36NH2. Its accuracy was evaluated from measurements of plasma obtained during intravenous infusions (1.5 pmol x kg-1 x min-1) of GLP-1 7-36NH2 in healthy subjects and patients with type 2 diabetes. Plasma levels of the endogenous GLP-1 metabolite increased during a meal challenge in patients with type 2 diabetes, and treatment with a DPP-4 inhibitor fully blocked its formation. Accurate measurements of the GLP-1 metabolite may contribute to understanding its physiology and role of GLP-1 in diabetes. [ABSTRACT FROM AUTHOR]

Background: Medical education is a cornerstone in the global combat against diseases such as diabetes and obesity which together affect more than 500 million humans. Massive Open Online Courses (MOOCs) are educational tools for institutions to teach and share their research worldwide. Currently, millions of people have participated in evidencebased MOOCs, however educational and professional benefit(s) for course participants of such initiatives have not been addressed sufficiently. We therefore investigated if participation in a 6 week open online course in the prevention and treatment of diabetes and obesity had any impact on the knowledge, skills, and career of health care professionals contrasting participants from developing countries versus developed countries. Methods: 52.006 participants signed up and 29.469 participants were active in one of the three sessions (2014-2015) of Diabetes - a Global Challenge. Using an online based questionnaire (nine sections) software (Survey Monkey), email invitations were send out using a Coursera based database to the 29.469 course participants. Responses were analyzed and stratified, according to the United Nations stratification method, by developing and developed countries. Results: 1.303 (4.4%) of the 29.469 completed the questionnaire. 845 of the 1303 were defined as health care professionals, including medical doctors (34%), researchers (15%), nurses (11%) and medical students (8%). Over 80% of the health care participants report educational benefits, improved knowledge about the prevention and treatment therapies of diabetes and furthermore improved professional life and practice. Over 40% reported that their professional network expanded after course participation. Study participants who did not complete all modules of the course reported similar impact as the ones that completed the entire course(P = 0.9). Participants from developing countries gained more impact on their clinical practice (94%) compared to health care professionals from developed regions (88%) (Mean of differences = 6%, P = 0.03. Conclusions: Based on self-reports from course participants, MOOC based medical education seems promising with respect to providing accessible and free research-based education to health professionals in both developing and developed countries. Course participants from developing countries report more benefits from course participation than their counterparts in the developed world. [ABSTRACT FROM AUTHOR]

Glucagon levels are increasingly being included as endpoints in clinical study design and more than 400 current diabetes-related clinical trials have glucagon as an outcome measure. The reliability of immune-based technologies used to measure endogenous glucagon concentrations is, therefore, important. We studied the ability of immunoassays based on four different technologies to detect changes in levels of glucagon under conditions where glucagon levels are strongly suppressed. To our surprise, the most advanced technological methods, employing electrochemiluminescence or homogeneous time resolved fluorescence (HTRF) detection, were not capable of detecting the suppression induced by a glucose clamp (6 mmol/L) with or without atropine in five healthy male participants, whereas a radioimmunoassay and a spectrophotometry-based ELISA were. In summary, measurement of glucagon is challenging even when state-of-the-art immune-based technologies are used. Clinical researchers using glucagon as outcome measures may need to reconsider the validity of their chosen glucagon assay. The current study demonstrates that the most advanced approach is not necessarily the best when measuring a low-abundant peptide such as glucagon in humans. [ABSTRACT FROM AUTHOR]

Glucagon circulates in concentrations in the low picomolar range, which is demanding regarding the sensitivity of the methods for quantification applied. In addition, the differential and tissue specific proteolytic processing of the glucagon precursor and the presence in of several glucagon-like sequences, not only in the precursor of glucagon, but also in a number of other peptides of the glucagon-secretin family of peptides, put special demands on the specificity of the assays. Finally, experience has shown that unspecific interference of plasma components has presented additional problems. All of these problems have resulted in a lot of diverging results concerning measured and reported glucagon responses in both humans and experimental animals that have and still are causing considerable debate and controversy. There is very solid evidence that glucagon is an important hormone in human and mammalian metabolism, but its precise physiological role in glucose and lipid metabolism and in metabolic disease has been difficult to establish, not least because of these difficulties. It was our purpose with this review to discuss the methods of glucagon quantification and discuss pitfalls and sources of error. We also reviewed some of the dogmas regarding glucagon secretion in the light of the methodological difficulties. [ABSTRACT FROM AUTHOR]

Hundred years after the discovery of glucagon, its biology remains enigmatic. Accurate measurement of glucagon has been essential for uncovering its pathological hypersecretion that underlies various metabolic diseases including not only diabetes and liver diseases but also cancers (glucagonomas). The suggested key role of glucagon in the development of diabetes has been termed the bihormonal hypothesis. However, studying tissue-specific knockout of the glucagon receptor has revealed that the physiological role of glucagon may extend beyond blood-glucose regulation. Decades ago, animal and human studies reported an important role of glucagon in amino acid metabolism through ureagenesis. Using modern technologies such as metabolomic profiling, knowledge about the effects of glucagon on amino acid metabolism has been expanded and the mechanisms involved further delineated. Glucagon receptor antagonists have indirectly put focus on glucagon's potential role in lipid metabolism, as individuals treated with these antagonists showed dyslipidemia and increased hepatic fat. One emerging field in glucagon biology now seems to include the concept of hepatic glucagon resistance. Here, we discuss the roles of glucagon in glucose homeostasis, amino acid metabolism, and lipid metabolism and present speculations on the molecular pathways causing and associating with postulated hepatic glucagon resistance. [ABSTRACT FROM AUTHOR]