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Glucagon and glucagon-like peptide-1 (GLP-1) have important roles in the development and treatment of diabetes. Their plasma concentrations are currently being measured in more than 500 clinical studies according to clinicaltrial.gov. As glucagon and total GLP-1 (reflecting the secretion and action of GLP-1 and is analytically independent of DPP-4 activity) circulate in the low picomolar range it has been troublesome to accurately measure their plasma concentrations. Equally important are so-called preanalytical considerations including the use of enzyme inhibitors. The latter may also depend on the analytical approach, for example single-antibody assay versus sandwich ELISA. To provide guidelines for measurement of glucagon and GLP-1 in clinical trials we obtained blood samples using four different blood-containers (with either neprilysin inhibitor: sacubitrilat 25 µg/mL; trasylol 500KIU/mL; P800 tubes; or vehicle) from healthy individuals (male/female; 7/4; mean ± SD; age: 32 ± 12 years, BMI; 26 ± 4 kg/m2) after an overnight fast and following an intravenous amino acid (AA) infusion (14 g/L; 331 mg/min/kg body weight. Plasma concentrations of glucagon increased significantly during the AA-infusion independent of blood containers and assays. The sandwich ELISA showed significant lower levels of glucagon with addition of neprilysin inhibitor whereas both trasylol and P800 tubes resulted in increased levels during the AA-infusion. Glucagon levels measured by the two radioimmunoassays (RIA) did not depend on the containers. Levels of total GLP-1 did not depend on the containers using a sandwich ELISA (Mercodia) but increased by a factor of a 2.5 when plasma from P800 containers was assayed using a C-terminal specific RIA. In summary, levels of glucagon and GLP-1 depend on both preanalytical and analytical factors that should be standardized for future clinical studies. Disclosure S. Kjeldsen: None. N.J. Jensen: None. N. Heinz: None. B. Hartmann: Board Member; Bainan Biotech. J.J. Holst: Advisory Panel; Novo Nordisk. Board Member; Antag Therapeutics, Bainan Biotech. N.J. Wewer Albrechtsen: Research Support; Mercodia AB, Novo Nordisk, Regeneron Pharmaceuticals Inc. Speaker's Bureau; Merck & Co., Inc., Mercodia AB. Funding Nicolai J. Wewer Albrechtsen were financed by NNF Excellence Emerging Investigator Grant – Endocrinology and Metabolism (Application No. NNF19OC0055001) , EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude.

Glucagon is essential for glucose control and increased levels of glucagon (hyperglucagonemia) observed in patients with type 2 diabetes contribute to their hyperglycemia. Recently, hyperglucagonemia has also been found in individuals with non-alcoholic fatty liver disease (NAFLD) as well as impaired actions of glucagon on amino acid catabolism. Whether glucagon actions on hepatic glucose production are impaired is unknown. We investigated the acute effects of a single bolus of glucagon (0.2mg) on glucose dynamics in 18 normoglycemic individuals (age: 51±3 years, BMI; 31± 0.8kg/m2, hepatic fat content: 20±2%, fasting glucose: 5.5±0.1mM) with magnetic resonance imaging verified NAFLD and 22 controls (age: 38±3 years, BMI; 24± 0.8kg/m2, hepatic fat content: 4±0.1%, fasting glucose: 5.0±0.1mM) . On a separate day, a mixture of amino acids (14 g/L; 331 mg/min/kg body weight) was infused intravenously for 45min to evaluate the actions of endogenous glucagon on glucose dynamics. Glucose levels (see figure) were significantly increased in individuals with NAFLD 60min after the glucagon bolus and during the amino acid infusion with a maximal difference of 0.5mM 30min into the infusion. These data suggest that the actions of glucagon on hepatic glucose production are not impaired by NAFLD. Therefore, the hyperglucagonemia in patients with NAFLD may constitute a diabetogenic risk factor. Disclosure S.Kjeldsen: None. H.Vilstrup: None. F.V.Schiødt: Advisory Panel; Novo Nordisk. A.Møller: None. E.B.Rashu: None. L.Gluud: Advisory Panel; Novo Nordisk, Consultant; Pfizer Inc., Research Support; Alexion Pharmaceuticals, Inc., Gilead Sciences, Inc., Novo Nordisk, Sobi. S.B.Haugaard: None. J.J.Holst: Advisory Panel; Novo Nordisk, Board Member; Antag Therapeutics, Bainan Biotech. J.Rungby: Advisory Panel; Abbott, Boehringer Ingelheim International GmbH, Speaker’s Bureau; AstraZeneca, Bayer AG, Novo Nordisk, Pfizer Inc. N.J.Wewer albrechtsen: Research Support; Mercodia AB, Novo Nordisk, Regeneron Pharmaceuticals Inc., Speaker’s Bureau; Merck & Co., Inc., Mercodia AB. N.J.Jensen: None. M.Nilsson: None. N.Heinz: None. J.D.Nybing: None. F.H.Linden: None. E.Høgh-schmidt: n/a. M.P.Boesen: None. S.Madsbad: None. Funding NNF Excellence Emerging Investigator Grant – Endocrinology and Metabolism (Application No. NNF19OC0055001) , EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude.