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Amino acids control alpha cell secretion and growth, while glucagon stimulates hepatic amino acid uptake and ureagenesis, forming the liver-alpha cell axis. Patients with nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes have increased levels of glucagon and amino acids pointing to a disrupted liver-alpha cell axis. Both glucagon agonism and antagonism are explored for diabetes and NAFLD therapy but their effects on the axis are unclear. Female C57Bl/6JRj mice were treated with a long-acting glucagon analogue (GCGA, NNC9204-0043, Novo Nordisk A/S) or PBS twice daily, or once weekly with a GCGR antibody (GCGR Ab, REGN1193, Regeneron) or control antibody (Ctl. Ab, REGN1945, Regeneron) . After four weeks, total plasma amino acids were decreased by 48% in GCGA mice (P=0.0006) and increased by 285% in GCGR Ab mice (P Disclosure E. E. Christensen: None. 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. K. D. Galsgaard: None. C. D. Johansen: None. S. Trammell: None. A. B. Bomholt: None. J. Hunt: None. T. Kruse: Employee; Novo Nordisk. J. F. Lau: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. T. Grevengoed: None. Funding Nicolai J. Wewer Albrechtsen is supported by NNF Excellence Emerging Investigator Grant ? Endocrinology and Metabolism (Application No. NNF19OC0055001) , EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude (1052-00003B) . Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (grant agreement NNF14CC0001) .

Metabolic dysfunction in patients with metabolic fatty liver disease (MAFLD) may increase the risk for diabetes development. The liver is essential for the postprandial control of our metabolism and hormonal response, yet most studies focus on fasting conditions. We therefore studied the fasting and postprandial phase in individuals with biopsy-proven nonalcoholic fatty liver disease (NAFLD, (n = 9, mean age 50 y, mean BMI 35 kg/m2, no/mild fibrosis) , cirrhosis (n = 10, age 62 y, BMI 32 kg/m2, CHILD A/B) and healthy controls (n = 10, age 23, BMI 25 kg/m2) , randomized 1:1 to fasting or Nutridrink (Nutricia, 300 kcal) . None of the postprandial patients had type 2 diabetes (T2D) . In the NAFLD/cirrhosis groups, 17/classified as MAFLD. Peripheral blood samples were collected at 0, 15, 45, 60, 90 and 120 minutes. At time point 60, a transjugular liver biopsy and liver vein blood were taken. Levels of glucose, insulin, C-peptide, glucagon, and fibroblast growth factor 21 (FGF21) were measured in peripheral blood, glucagon and FGF21 also in liver vein blood. Postprandial peak glucose and C-peptide was increased in NAFLD and cirrhosis compared with healthy (mean peak glucose (mM) 7, 10, 6; mean peak C-peptide (pM) 2675, 3340, 1689, respectively) . The postprandial incremental AUC for insulin was significantly increased in patients with NAFLD compared to healthy. Patients with NAFLD and cirrhosis had hyperglucagonemia, a phenotype related to prediabetes. FGF21 was increased in NAFLD and cirrhosis and correlated to age (r= .61, P = .001) and fasting glucose (r = .54, P = .006) . Glucagon levels were higher in liver vein compared to peripheral blood, while FGF21 levels were similar in both compartments. In summary, patients with NAFLD and cirrhosis without T2D showed significant metabolic dysfunction after a standardized meal compared to healthy controls. We found impaired glucose tolerance, hyperinsulinemia and hyperglucagonemia in both NAFLD and cirrhosis, suggesting a condition of prediabetes in patients with MAFLD. Disclosure J. Grandt: None. A.H. Jensen: None. M.P. Werge: None. E.B. Rashu: None. A. Junker: None. L. Hobolth: None. C. Mortensen: None. M. Vyberg: None. R. Serizawa: Consultant; Merck Sharp & Dohme Corp. L. Gluud: Advisory Panel; Novo Nordisk. Consultant; Pfizer Inc. Research Support; Alexion Pharmaceuticals, Inc., Gilead Sciences, Inc., Novo Nordisk, Sobi. 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 was financed by NNF Excellence Emerging Investigator Grant – Endocrinology and Metabolism (Application No. NNF19OC0055001) , EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude.

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.

Patients with nonalcoholic fatty liver disease (NAFLD) have elevated fasting levels of glucagon (gcg) and amino acids (AA) . Gcg regulates hepatic AA catabolism by augmenting ureagenesis. We hypothesized that hepatic steatosis causes gcg resistance and impaired gcg-induced hepatic ureagenesis. To address this, we developed an isolated liver perfusion model in mice and used it to investigate gcg’s direct effect on hepatic glucose production (HGP) and ureagenesis in 9 male mice with hepatic steatosis (NAFLD mice) (B6.Vlepob/ob/JRj) and healthy controls (CON) (C57bl6/JRj) . NAFLD mice had higher body mass (50 vs. 26 g, P Disclosure F.R.Ceutz: None. E.E.Christensen: None. M.Winther-soerensen: None. H.Vilstrup: None. 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.

Gluco-regulatory disturbances such as hepatic insulin resistance, hyperinsulinemia and prediabetes are commonly present in patients with nonalcoholic fatty liver disease (NAFLD) and those individuals may over time develop full-blown type 2 diabetes. Chronic liver diseases­ such as NAFLD and autoimmune liver diseases (AILDs) are heterogenous but may affect glucose-metabolism similarly. It is, however, unknown if AILDs—such as primary biliary cholangitis (PBC) —display gluco-regulatory impairments. We therefore investigated glucose and hormonal responses during a 75 g oral glucose tolerance test (OGTT) in patients with biopsy-verified, non-cirrhotic PBC (n = 9, age 55 ± y (mean ± sd) , BMI 31 ± 6 kg/m2 (mean ± sd)) , NAFLD (n = 6, age 38 ± 17 y, BMI 31 ± 4 kg/m2) and healthy controls (n = 8, age 23 ± 3 y, BMI 23 ± 2 kg/m2) . None of the participants had diabetes. In the PBC group, 3 had NAFLD. Fasting glucose, c-peptide and insulin levels were significantly increased in PBC and NAFLD compared with healthy controls ([mean (95 % CI) ]; glucose (mM) 5.6 (4.7-6.7) , 5.7 (5.2-6.1) , 4.7 (3.9-5.6) ; c-peptide (pM) 993 (556-1773) , 1334 (1036-1719) , 483 (268-869) ; insulin (pM) 98 (33-298) , 166 (103-267) , 43 (14-136) ; respectively) . Hepatic insulin resistance (reflected by fasting homeostasis model assessment of insulin resistance (HOMA-IR)) was present in PBC (mean 4.0 (95 % CI 1.2-13.9)) and NAFLD (7.0 (4.1-11.9)) but not in healthy controls (1.5 (0.4-5.4)) . There was no significant difference in glucose levels between the groups. Beta-cell secretion (c-peptide) was significantly increased in PBC and NAFLD. Insulin responses were higher in PBC and NAFLD compared with healthy but only reached statistical significance in NAFLD. Our data suggest that patients with PBC have gluco-regulatory disturbances including hepatic insulin resistance and impaired beta-cell function. Metabolic dysfunction of PBC may be underestimated and warrant further investigation. Disclosure A.H. Jensen: None. H. Ytting: Other Relationship; Gilead Sciences, Inc. J. Grandt: None. M.P. Werge: None. E.B. Rashu: None. L.E. Hetland: None. A. Junker: None. L. Hobolth: None. C. Mortensen: None. F. Tofteng: None. M. Vyberg: None. R. Serizawa: Consultant; Merck Sharp & Dohme Corp. L. Gluud: Advisory Panel; Novo Nordisk. Consultant; Pfizer Inc. Research Support; Alexion Pharmaceuticals, Inc., Gilead Sciences, Inc., Novo Nordisk, Sobi. 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.

Glucagon receptor antagonists (GRAs) reduce blood glucose in subjects with type 2 diabetes (T2D) but may cause dyslipidemia. Increased plasma triglyceride (TG) levels in patients with T2D are thought to be caused partly by insulin resistance but it is unknown if altered glucagon receptor (GCGR) signaling contributes. We investigated the effects of chronic GCGR inhibition and activation on plasma TG and cholesterol levels during an 180 min oral lipid tolerance test (µL/g body weight olive oil; OLTT) in GCGR knockout (Gcgr-/-) mice, and in C57Bl/6JRj mice treated with a GCGR antibody (GCGR Ab, REGN1193, Regeneron) or a long-acting glucagon analogue (GCGA, NNC9204-0043, Novo Nordisk A/S) . Plasma TG levels increased 3-fold in Gcgr-/- mice compared to wild-type littermates (Gcgr+/+) during the OLTT. Eight weeks treatment with GCGR Ab increased plasma TG levels during OLTTs by 39% whereas GCGA reduced levels by 39%. Low-density lipoprotein levels, estimated using fast protein liquid chromatography, were increased in Gcgr-/- mice and GCGR Ab treated mice, whereas GCGA treatment lowered very-low density lipoprotein levels. We next investigated the effects of acute GCGR inhibition and activation; a single treatment with GRA (25-2648, Novo Nordisk A/S) and GCGA respectively increased and decreased TG levels by 55% and 32% during OLTTs. Our study suggests that impaired and increased GCGR signaling respectively impairs and accelerates TG metabolism, chronically and acutely, in female mice. We were unable to identify GCGR in adipocytes using autoradiography and immunohistochemistry and our data thus implicate that glucagon has acute and chronic effects on intestinal lipid uptake and/or acts to enhance hepatic lipid metabolism in mice. Thus, impaired actions of glucagon may cause hypertriglyceridemia, while enhancing GCGR signaling may benefit subjects with dyslipidemia. Disclosure K.D.Galsgaard: None. E.E.Christensen: None. A.B.Bomholt: None. J.Hunt: None. T.Kruse: Employee; Novo Nordisk. J.F.Lau: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. C.Christoffersen: None. 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 This work was supported by the Novo Nordisk Foundation (NNF) Center for Basic Metabolic Research University of Copenhagen, (NNF Application Number: 13563) ; The A.P. Møller Foundation; NNF Tandem Programme (NNF Application Number: 31526) ; NNF Project Support in Endocrinology and Metabolism–Nordic Region (NNF Application Number: 34250) . 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. Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (grant agreement NNF14CC0001) .

Glucagon secretion is regulated by circulating glucose, but it has turned out that amino acids also play an important role and that hepatic amino acid metabolism and glucagon are linked in a mutual feedback cycle, the liver–α-cell axis. On the basis of this knowledge, we hypothesized that hepatic steatosis might impair glucagon’s action on hepatic amino acid metabolism and lead to hyperaminoacidemia and hyperglucagonemia. We subjected 15 healthy lean and 15 obese steatotic male participants to a pancreatic clamp with somatostatin and evaluated hepatic glucose and amino acid metabolism when glucagon was at basal levels and at high physiological levels. The degree of steatosis was evaluated from liver biopsy specimens. Total RNA sequencing of liver biopsy specimens from the obese steatotic individuals revealed perturbations in the expression of genes predominantly involved in amino acid metabolism. This group was characterized by fasting hyperglucagonemia, hyperaminoacidemia, and no lowering of amino acid levels in response to high levels of glucagon. Endogenous glucose production was similar between lean and obese individuals. Our results suggest that hepatic steatosis causes resistance to the effect of glucagon on amino acid metabolism. This results in increased amino acid concentrations and increased glucagon secretion, providing a likely explanation for fatty liver–associated hyperglucagonemia.

Hyperglucagonemia is a common observation in both obesity and type 2 diabetes, and the etiology is primarily thought to be hypersecretion of glucagon. We investigated whether altered elimination kinetics of glucagon could contribute to the hyperglucagonemia in type 2 diabetes and obesity. Individuals with type 2 diabetes and preserved kidney function (8 with and 8 without obesity) and matched control individuals (8 with and 8 without obesity) were recruited. Each participant underwent a 1-hour glucagon infusion (4 ng/kg/min), achieving steady-state plasma glucagon concentrations, followed by a 1-hour wash-out period. Plasma levels, the metabolic clearance rate (MCR), half-life (T½) and volume of distribution of glucagon were evaluated and a pharmacokinetic model was constructed. Glucagon MCR and volume of distribution were significantly higher in the type 2 diabetes group compared to the control group, while no significant differences between the groups were found in glucagon T½. Individuals with obesity had neither a significantly decreased MCR, T½, nor volume of distribution of glucagon. In our pharmacokinetic model, glucagon MCR associated positively with fasting plasma glucose and negatively with body weight. In conclusion, our results suggest that impaired glucagon clearance is not a fundamental part of the hyperglucagonemia observed in obesity and type 2 diabetes.

Glucagon regulates metabolic functions through the glucagon receptor (GCGR). Agonists as well as antagonists of GCGR are evaluated as treatments of diabetes and obesity. To understand the role of glucagon, knowledge of GCGR localization is important but limited. Twelve commercially available GCGR antibodies were evaluated using HEK293 cells transfected with mouse or human GCGR cDNA transcripts. Eleven of the 12 GCGR antibodies showed staining of GCGR protein from both species, but not in cells transfected with a scrambled vector. Several of the antibodies stained liver sections from both GCGR+/+ and GCGR-/- mice. Staining was absent in fat and muscle tissues but present in the distal tubuli of the kidney. Two antibodies did not react with tissue from GCGR-/- and were further evaluated by western blotting of GCGR transfected cells. Bands corresponding to the predicted size of the GCGR (62kDa) were identified but larger bands were also observed. Liver tissue from healthy subjects and individuals with nonalcoholic steatohepatitis (NASH) was stained using same GCGR antibodies and based on qualitative intensities GCGR staining did not seem to vary between healthy and NASH liver sections. We used autoradiography to confirm the immunohistochemistry findings. In mice injected with 125I-labeled glucagon, grains were found in the liver and in the distal tubuli of the kidney, whereas fat and muscle sections were negative. To investigate the role of renal GCGR’s, we infused glucagon (1µg) in rats with and without blockade of the GLP-1 receptor (GLP-1R) (Exendin 9-39). Glucagon increased diuresis and renal blood flow, but not blood pressure, in a GLP-1R dependent manner. Urea excretion was numerically increased by glucagon (P=0.08). In conclusion, glucagon receptor localization is challenging due to the lack of suitable commercially available GCGR antibodies. The actions of glucagon on renal parameters may be mediated by the GLP-1R. Glucagon’s role in kidney physiology needs further investigation. Disclosure S. Kjeldsen: None. K.D. Galsgaard: None. M. Winther-Soerensen: None. R. Serizawa: None. J. Pedersen: None. C. Orskov: None. R. Albrechtsen: None. C.M. Sorensen: None. L. Gluud: None. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. Funding Novo Nordisk Foundation (NNF19OC0055001)

In type 1 diabetes (T1D), the counterregulatory glucagon response to hypoglycemia disappears over time. It remains unknown whether this is due to lack of glucagon production or represents a malfunctioning stimulus-secretion coupling in pancreatic alpha cells. Arginine test (5 g of arginine infused intravenously (iv) over 1 minute after an overnight fast) with frequent measurements of plasma glucagon and serum C-peptide concentrations over 30 minutes was performed in 80 patients with T1D (19/61 females/males, age 49 (22-74) years (median (range)), duration 20 (2-46) years, BMI 28 (22-44) kg/m2, HbA1c 8.2 (7.5-10) %). The interrelationship between the arginine-induced glucagon response and demographic, clinical and biochemical parameters was evaluated using a stepwise approach in two multiple linear regression analysis models with glucagon area under curve (AUC) and Cmax, respectively, as dependent parameter. Mean baseline plasma glucagon concentrations amounted to 10.6±5.5 pmol/L (mean±SD). Overall, robust arginine-induced glucagon responses were observed (AUC: 499±209 pmol/L × min; Cmax: 41.0±17.7 pmol/L) independently of diabetes duration, age, HbA1c and beta cell secretory capacity. Fasting plasma glucagon and waist-to-hip ratio predicted the glucagon response in both models. When leaving out fasting plasma glucagon of the models, fasting low-density lipoprotein cholesterol and alanine aminotransferase as well as eGFR also predicted the glucagon response. In conclusion, glucagon secretory capacity in patients with T1D as assessed by iv arginine test seems preserved independently of diabetes duration, age, HbA1c and beta cell secretory capacity positioning pharmaceutical restoration of the glucagon counterregulatory response to hypoglycemia as an attractive therapeutic strategy for the protection of insulin-induced hypoglycemia. Disclosure J. Warnøe: None. T. F. Dejgaard: Advisory Panel; Self; Novo Nordisk, Consultant; Self; Boehringer Ingelheim International GmbH, Research Support; Self; AstraZeneca, Novo Nordisk, Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk. F. K. Knop: Advisory Panel; Self; MSD Corporation, Novo Nordisk A/S, Sanofi, Consultant; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Novo Nordisk A/S, Pharmacosmos, Zealand Pharma A/S, Research Support; Self; Novo Nordisk A/S, Zealand Pharma A/S, Speaker's Bureau; Self; AstraZeneca, Bayer AG, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, MSD Corporation, Novo Nordisk A/S. N. J. Johansen: Employee; Self; Novo Nordisk. A. Lund: Speaker's Bureau; Self; Novo Nordisk, Sanofi. J. I. Bagger: None. N. J. Wewer albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Regeneron Pharmaceuticals Inc., Speaker's Bureau; Self; Merck & Co., Inc., Mercodia. B. Hartmann: None. J. J. Holst: Consultant; Self; Novo Nordisk, Other Relationship; Self; Antag Therapeutics, Bainan Biotech, MSD Corporation, Novo Nordisk, Other Relationship; Spouse/Partner; Antag Therapeutics, Bainan Biotech, Synklino ApS. T. Vilsbøll: Consultant; Self; Amgen Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Bristol-Myers Squibb Company, Gilead Sciences, Inc., Lilly Diabetes, Merck Sharp & Dohme Corp., Mundipharma International, Novo Nordisk, Sanofi, Sun Pharmaceutical Industries Ltd. H. U. Andersen: Stock/Shareholder; Self; Novo Nordisk A/S.

Glucagon regulates hepatic glucose production and hyperglucagonemia contributes to diabetes. Equally important, glucagon may regulate amino acid (AA) levels that in turn control glucagon secretion. Hepatic steatosis may uncouple glucagon’s effect on AA metabolism causing impaired actions of glucagon (resistance) on AA metabolism but not glucose production, thereby creating a diabetogenic circle. In order to quantify glucagon’s effect on AA metabolism, we developed and evaluated a glucagon sensitivity test. The test consists of a bolus-infusion of glucagon (200 μg) and an AA infusion (330 mg/min/kg body weight for 45 min) on two separate days following an overnight fast. Liver fat was measured using magnetic resonance imaging. Preliminary data from six individuals without diabetes (HbA1c Disclosure S. Kjeldsen: None. E. B. Rashu: None. L. Gluud: None. S. B. Haugaard: None. J. J. Holst: Consultant; Self; Novo Nordisk, Other Relationship; Self; Antag Therapeutics, Bainan Biotech, MSD Corporation, Novo Nordisk, Other Relationship; Spouse/Partner; Antag Therapeutics, Bainan Biotech, Synklino ApS. J. Rungby: None. N. J. Wewer albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Regeneron Pharmaceuticals Inc., Speaker’s Bureau; Self; Merck & Co., Inc., Mercodia. N. J. Jensen: None. M. Nilsson: None. J. D. Nybing: None. F. H. Linden: None. E. Hogh-schmidt: None. M. P. Boesen: None. S. Madsbad: Advisory Panel; Self; AstraZeneca; Boehringer Ingelheim; Eli Lilly; Merck Sharp & Dohme; Novo Nordisk; Sanofi, Research Support; Self; Novo Nordisk, Boehringer Ingelheim, Speaker’s Bureau; Self; AstraZeneca; Boehringer Ingelheim; Merck Sharp & Dohme; Novo Nordisk; Sanofi. H. Vilstrup: None. Funding Novo Nordisk Foundation (116113)

Glucagon (gcg) regulates hepatic glucose and amino acid (AA) metabolism, and increased gcg levels (hyperglucagonemia) contribute to hyperglycemia in diabetes. We hypothesized that the enzyme neprilysin (NEP) contributes to gcg degradation. We measured plasma gcg levels during a mixed meal in nine healthy men after a single dose of the NEP-inhibitor/angiotensin II receptor blocker (194 mg sacubitril/206 mg valsartan, 30 min prior to meal), a DPP-4 inhibitor (sitagliptin, 2x100mg), these combined, or the meal alone. Postprandial gcg levels were 2.7-fold higher in sacubitril/valsartan treated individuals compared to controls (P=0.005) and this was not significantly altered by the addition of sitagliptin (P=0.28). Sacubitril/valsartan also lowered postprandial plasma AA levels (P=0.01), but glucose levels were unaffected (P=0.76). In obese individuals (n=7), eight weeks sacubitril/valsartan treatment increased fasting gcg levels (P=0.02). To test whether NEP degrades gcg and diminishes its signaling, we performed mass-spectrometry and assessed cells transfected with the gcg receptor (gcgr). We found that NEP cleaves gcg and that the gcg fragments produced were unable to activate the gcgr. In non-sedated C57BL/6JRj female mice (n=8) NEP inhibition (sacubitril, 0.7 nmol/g) increased gcg levels (P=0.02) and tended to increase AA disappearance (P=0.076) and urea formation (P=0.08) during an AA challenge. A gcgr antagonist (Novo Nordisk; 25-2648, 100 mg/kg) abolished the increase in urea formation observed with sacubitril alone. Gcg levels were increased 1.9-fold (P Disclosure S. Kjeldsen: None. S. Zraika: Research Support; Self; Novartis Pharmaceuticals Corporation. S.M. Mongovin: None. L.H. Hansen: None. D. Terzic: None. P.D. Mark: None. P. Plomgaard: None. J.P. Gøtze: None. M. Winther-Soerensen: None. J. Hunt: None. K.D. Galsgaard: None. M.M. Rosenkilde: Board Member; Self; Synklino. Consultant; Self; Antag Therapeutics, Bainan Biotech. G.H. Goossens: None. E.E. Blaak: None. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. Funding Novo Nordisk Foundation (NNF19OC0055001)

Introduction: Glucagon plays a role in the pathogenesis of type 2 diabetes. Yet, little is known about the effect of interventions aimed at preventing progression from prediabetes to diabetes on glucagon concentrations. We examined the effects of dapagliflozin, metformin or exercise on plasma glucagon concentration in individuals with HbA1c-defined prediabetes. Methods: 120 individuals were randomized to a 13-week intervention with dapagliflozin (10 mg once daily), metformin (850 mg twice daily), exercise (interval training 30 min 5 days/week) or control (habitual living). A 75 g oral glucose tolerance test (OGTT; 0, 30, 60 and 120 min) was administered at baseline, at 13 weeks (end of intervention) and at 26 weeks (end of follow-up). Linear mixed-effects models were used to assess effects on fasting concentration, early (0-30 min) area under the curve relative to the fasting level (rAUC0-30 min) and full rAUC0-120 min for glucagon. Results: At baseline, the median (Q1;Q3) age was 62 (54;68) years, HbA1c 5.9% (5.7;6.1%), fasting glucagon 11 (7;15) pmol/L, and 56% were men. No differences in the glucagon measures between groups from baseline to 13 or 26 weeks were observed (Table 1). Conclusions: 13 weeks of treatment with dapagliflozin, metformin or exercise was not associated with changes in fasting or post-OGTT glucagon concentrations in individuals with prediabetes. Disclosure K.K.B. Clemmensen: Research Support; Self; AstraZeneca, Novo Nordisk Foundation. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. M.B. Blond: None. H. Amadid: None. L.B. Nielsen: None. D. Vistisen: Stock/Shareholder; Self; Novo Nordisk A/S. K. Karstoft: Employee; Spouse/Partner; Novo Nordisk A/S. F. Persson: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk A/S. Research Support; Self; Amgen, AstraZeneca, Novo Nordisk A/S. Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Merck Sharp & Dohme Corp., Mundipharma International, Novo Nordisk A/S. M. Ried-Larsen: None. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. S.S. Torekov: Research Support; Self; Novo Nordisk Inc. J.S. Quist: None. M.E. Jørgensen: Research Support; Self; Amgen, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Sanofi-Aventis. Stock/Shareholder; Self; Novo Nordisk A/S. K. Færch: None. Funding Novo Nordisk Foundation; AstraZeneca; University of Copenhagen; Innovation Foundation

Plasma concentrations of the two pancreatic enzymes, amylase and lipase, are increased within the normal physiological range after initiation of glucagon-like peptide-1 receptor agonist (GLP-1RA) treatment. An association between the use of GLR-1RA and incidence of acute pancreatitis or pancreatic cancer has not been found - neither in rodent studies nor in preclinical studies or in the large cardiovascular outcome trials with GLP-1RAs. The increased concentrations of pancreatic enzymes have been suggested to be explained by increased DNA or protein synthesis found in rodent and acinar cell studies. However, whether this translates into humans is unknown. We therefore investigated the effect of liraglutide, a GLP-1RA, on pancreatic volume, edema and DNA synthesis reflected by 18F-flourothymidine (FLT)-uptake using state-of-the-art positron emission tomography (PET)-magnetic resonance imaging (MRI) before initiation, after four weeks during titration of liraglutide and after six weeks during steady state on maximum dose of liraglutide 3.0 mg in 14 obese men (age 38 ± 3 years, BMI 32 ± 1 kg/m2) without diabetes. Plasma concentrations of amylase and lipase were assessed in parallel. Amylase (+7 U/L [95% confidence intervals, 3 - 11] p In conclusion, six weeks of treatment with liraglutide did not affect pancreatic volume, edema or cellularity in obese individuals. Increased DNA synthesis reflected by FLT-uptake in the pancreas seen after four weeks of liraglutide treatment may be responsible for the increase in pancreatic enzymes. Disclosure M.S. Svane: None. H.H. Johannesen: None. C. Martinussen: None. K.N. Bojsen-Moller: None. M.L. Hansen: None. A.E. Hansen: None. C.F. Deacon: Employee; Spouse/Partner; Merck & Co., Inc. Stock/Shareholder; Spouse/Partner; Merck & Co., Inc. Other Relationship; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck & Co., Inc., Novartis AG, Novo Nordisk A/S. B. Hartmann: None. S.H. Keller: None. T.L. Klausen: None. A. Loft: None. A. Kjaer: None. S. Madsbad: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi-Aventis. Research Support; Self; Boehringer Ingelheim International GmbH, Novo Nordisk A/S. Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk A/S. J.O. Löfgren: None. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp.

Objective: Congenital sucrase-isomaltase deficiency is prevalent in Arctic populations. Recently, a frameshift mutation, c.273-274delAG, in the sucrose-isomaltase encoding gene (SI) was identified. This mutation was predicted to cause complete loss of enzymatic function, and thus is a diagnostic marker for Congenital sucrase-isomaltase deficiency. We aimed to characterize the metabolic health among adult Greenlanders carrying the c.273-274delAG mutation. Material and Methods: We genotyped the c.273-274delAG mutation in two cohorts of Greenlanders comprising 4600 (Cohort I) and 1500 participants (Cohort II), respectively. We assessed the effect of the variant on markers of metabolic health. Results: In cohort I, homozygous carriers of the c.273-274delAG mutation had a markedly healthier metabolic profile than the remaining population, including lower BMI (beta, -2.0 kg/m2, p=3.1x10-5), fat% (-3.3 %, p=0.0004), weight (-4.8 kg, p=0.0005), and levels of triglycerides (-0.27 mmol/l, p=2.3x10-6) and remnant cholesterol (-0.11 mmol/l, p=4.2x10-5). Even though homozygous carriers had a significantly lower consumption of sugar, this did not fully explain the healthier phenotype in these individuals. In cohort II, metabolomics data revealed that particularly HDL metabolism was altered among homozygous carriers of the mutation, and that these individuals had markedly increased levels of circulating acetate (1.78 mmol/l, p=2.1x10-26). Based on these findings, we hypothesize that the observed phenotype is caused by increased fermentation of undigested carbohydrates in the gut, leading to increased amounts of circulating acetate, which increases lipid oxidation and reduces storage of fat. Conclusion: Our results show, that homozygous c.273-274delAG mutation carriers have a markedly healthier metabolic profile, and indicate that SI constitutes a promising new drug target, with potential to prevent obesity and dyslipidemia. Disclosure M.E. Jørgensen: Research Support; Self; Amgen, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Sanofi-Aventis. Stock/Shareholder; Self; Novo Nordisk A/S. M.K. Andersen: None. L. Skotte: None. E. Jørsboe: None. N. Grarup: None. P. Bjerregaard: None. B. Soborg: None. O. Pedersen: None. B. Feenstra: Employee; Spouse/Partner; Novo Nordisk Inc. N.J.K. Færgeman: None. A. Koch: None. I. Moltke: None. T. Hansen: None. A. Albrechtsen: None.

Objective: Polypharmacotherapy shows superior efficacy compared to monotherapy in correcting obesity and its co-morbidities in preclinical studies and clinical trials. Female organisms have been traditionally neglected in this research potentially contributing to an increased rate of adverse advents in women. To address this disparity we herein determined the efficacy of our monomeric peptide with a balanced agonism at the receptors for glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) to correct obesity, glucose metabolism, nonalcoholic fatty liver disease (NAFLD) and dyslipidemia in both sexes of a mouse model for diet-induced obesity (DIO) by combining physiological treatment endpoints with plasma proteomic profiling (PPP), a new unbiased diagnostic tool for the efficacy and optimization of pharmacological interventions. Methods: We performed metabolic phenotyping along with PPP in body weight matched male and female DIO mice treated for 21 days with either PBS, the single GIP and GLP-1 monoagonists, or our GLP-1/GIP coagonist. Results: GLP-1R/GIPR coagonism improved obesity, glucose intolerance, NAFLD and dyslipidemia with superior efficacy in both male and female mice compared to monoagonist treatments. PPP revealed in both sexes broader changes of plasma proteins after GLP-1/GIP coagonist compared to monoagonist treatments, including established and novel biomarkers for systemic inflammation, NAFLD and atherosclerosis. Subtle sex-specific differences have been observed in metabolic phenotyping and PPP. Conclusions: We herein report enhanced efficacy of our GLP-1/GIP coagonist in both sexes relative to monoagonists for the treatment of metabolic disease. Wider sex-specific reductions of circulating proteins after GLP-1/GIP coagonist treatment may reflect additional metabolic benefits that are currently achieved exclusively after bariatric surgery. Disclosure S.M. Hofmann: Advisory Panel; Spouse/Partner; Novo Nordisk A/S. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. M.H. Tschöp: Advisory Panel; Self; ERX Pharmaceuticals, Novo Nordisk Foundation. A. Feuchtinger: None. M. Brielmeier: None. B. Finan: Employee; Self; Novo Nordisk A/S. R. DiMarchi: Employee; Self; Novo Nordisk Inc. M. Kleinert: None. S. Sachs: None. T.D. Müller: Research Support; Self; Novo Nordisk Inc., Sanofi-Aventis. K. Stemmer: None. M. Mann: None.

Glucagon plays an important role in hepatic glucose metabolism as well as protein/amino acid metabolism but detailed knowledge about glucagon’s role in hepatic autophagy is limited. We hypothesized that glucagon regulate hepatic protein metabolism partly through activation of AMP-kinase (AMPK) that in turn lead to inhibition of mammalian target of rapamycin complex1 (mTORC1) and activation of Uncoordinated-51 like autophagy activating kinase-1 (ULK1) resulting in increased autophagy. In order to test this hypothesis, we infused chronically catheterized awake mice with [13C5]-glutamine [0.225 mg/(kg-min)] for 120 min after which a second line was connected infusing somatostatin [4 µg/(kg-min)] and insulin [0.1 mU/(kg-min)], with (n=7) or without glucagon [10 ng/(kg-min)] (n=6) for 90 min. This raised plasma glucagon concentration ~18-fold compared to control. Glucagon infusion increased phosphorylation of hepatic: AMPKThr172, RaptorSer792, ULK1Ser555 (P=0.03, P=0.03, and P=0.06, respectively, compared to control), and resulted in hepatic Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II accumulation (P=0.02), a marker of autophagy, assessed by immunoblotting. Glucagon treatment also increased the 13C enrichment (m+3) in liver glucose (P In conclusion, our data suggest that glucagon promotes hepatic proteolysis and ureagenesis by AMPK-mediated suppression of mTOC1 activity, leading to increased hepatic autophagy. Disclosure K.D. Galsgaard: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, LLC, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Consultant; Self; Novo Nordisk A/S. Consultant; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Gilead Sciences, Inc., iMetabolic Biopharma Corporation. Other Relationship; Spouse/Partner; iMetabolic Biopharma Corporation. Other Relationship; Self; Maze Therapeutics. K. Petersen: Advisory Panel; Spouse/Partner; Merck Sharp & Dohme Corp. Research Support; Self; Merck Sharp & Dohme Corp., National Institute of Diabetes and Digestive and Kidney Diseases. Research Support; Spouse/Partner; National Institute of Diabetes and Digestive and Kidney Diseases. A. Nasiri: Employee; Spouse/Partner; Medtronic. G. Cline: None. X. Zhang: None. J. Lee: None. B.T. Hubbard: None.

Background: Altered meal-associated secretion of gut peptides is key for the metabolic changes and weight-loss observed after Roux-en-Y gastric bypass (RYGB). Parenteral administration of the gut peptide neurotensin (NT) reduces food intake in rodents, and, like glucagon-like peptide-1 and peptide YY, the secretion of NT is markedly increased after RYGB. We therefore investigated the effect of intravenous (IV) NT on ad libitum food intake and appetite sensations. Design: Using a double-blinded, randomized placebo-controlled design, NT (2.5pmol/kg/min) or saline was infused IV in healthy young men to obtain NT concentrations similar to NT levels observed postprandially after RYGB. Four visits were performed in random order, after an acclimatization visit, to evaluate the main outcomes - ad libitum food intake and appetite sensations (visual analogue scale (VAS) questionnaires). Blood samples were collected for plasma and serum analyses (including entero-pancreatic peptides and glucose). NT or saline was infused after a basal period (t= -60 to 0 min). On two study days (n=18), an ad libitum meal was served after one hour of infusion (t= 60 min) followed by blood sampling/VAS until t= 120 min (infusion discontinued). On two other study days (n=16), a liquid mixed meal was ingested after one hour of infusion (t = 60 min) followed by blood sampling and VAS until an ad libitum meal was served at t= 240 min (infusion discontinued at t=270min). Results: Food intake was similar on the ad libitum meal (t = 60 min) days - NT (4150 (3607-4696)kJ, mean (95% confidence interval) vs. saline (3820 (3250-4405) kJ, P=0.062 (paired t-test)) and on the LMM + ad libitum meal (t= 240 min) days - NT (4320 (3698-5008)kJ vs. saline (4250 (3739-4870)kJ, P=0.80). NT infusions did not influence appetite sensations or elicit gastrointestinal side effects. Conclusions: Despite obtaining NT concentrations similar to what is observed postprandially after RYGB, we did not observe any differences in appetite sensations or food intake. Disclosure S. Veedfald: None. C. Martinussen: None. M.S. Svane: None. T. Justinussen: None. M.G. Hindsø: None. N. Hedbäck: None. C.B. Christiansen: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. K.N. Bojsen-Moller: None. B. Hartmann: None. M. Fenger: None. J.F. Rehfeld: None. S. Madsbad: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi-Aventis. Research Support; Self; Boehringer Ingelheim International GmbH, Novo Nordisk A/S. Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk A/S. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. Funding Desirée og Niels Ydes Fond; Læge Sophus og hustru Olga Friis Legat; Beckett Fonden; Hørslev Fonden; Aase og Ejnar Danielsens Fond; Fabrikant Frands Køhler Nielsen og hustrus legat; Carl og Ellen Hertz’ Legat

Skeletal muscle insulin resistance is the earliest defect in type 2 diabetes (T2D), however, whether this represents a cell-autonomous defect or is secondary to changes in hormones and other circulating factors in vivo is unclear. To identify primary drivers of skeletal muscle insulin resistance in T2D without interference of these systemic factors, we have developed a “disease-in-a-dish” model by differentiating induced pluripotent stem cells (iPSCs) from T2D patients and controls into skeletal myoblasts (iMyos) and studied their function in vitro (n=8 subjects per group). We find that T2D iMyos retain multiple features of in vivo insulin resistance including altered insulin signaling downstream of the IRS/AKT pathway, impaired insulin-stimulated glucose uptake, and reduced mitochondrial oxidation. More importantly, using global phosphoproteomics we identify nearly 1,000 phosphorylation sites that were significantly increased or decreased (FDR < 0.05) by T2D status. While many of the observed signaling defects in T2D iMyos occurred inside the classical insulin signaling pathway and included dysregulation of Akt, mTOR and forkhead box transcription factors, over 85% of these changes occurred in basal protein phosphorylation. These included phosphorylation of proteins in many pathways not typically involved in insulin resistance, such as mRNA splicing and processing, gene transcription, chromatin remodeling and cytoskeleton dynamics. Motif analysis revealed enrichment of a multiplicity of kinases including ROCK, mTOR/S6K and PKCs with potential targets involved in insulin action, cytoskeleton and cell cycle, without changes in total protein content. This broad dysregulated phosphorylation network reveals a new dimension in the complexity of the cell-autonomous mechanisms underlying insulin resistance in T2D, as well as new targets for therapy or prevention of the disease. Disclosure T.M. Batista: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. J.R. Zierath: None. M. Mann: None. C. Kahn: Advisory Panel; Self; ERX Pharmaceuticals, Kaleido Biosciences. Consultant; Self; AntriaBio, Flagship Pioneering, Sana-Cobalt. Funding National Institutes of Health (R01DK031036, R01DK033201); Swedish Research Council (2015-00165); Novo Nordisk Foundation (NNF18CC0034900)

We have previously shown that totally pancreatectomized patients (PX) and patients with type 2 diabetes (T2D) exhibit hypersecretion of glucagon in response to oral glucose stimulation whereas intravenous glucose suppresses plasma concentrations of glucagon. Here, we evaluated the expression of the gene encoding glucagon (GCG), glucagon peptide content and density of glucagon-positive cells in mucosal biopsies from the upper gastrointestinal tract of PX patients, patients with T2D and nondiabetic controls. Eight PX patients, eight patients with T2D and eight sex, age and BMI-matched healthy controls underwent upper enteroscopy with intestinal biopsy sampling followed by an intraluminal infusion of 100 ml 50% (w/v) glucose solution with frequent blood sampling. The mucosal biopsies were subjected to mRNA sequencing, immunohistochemistry using a new glucagon-directed C-terminal monoclonal antibody, peptide extraction and subsequent measurements of tissue concentrations of glucagon and other proglucagon-derived peptides. GCG expression in the small intestine was 3 to 4-fold higher in PX and T2D patients compared to controls (P Disclosure M. Baekdal: None. A.B. Lund: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp. J.I. Egholk: None. T. Jorsal: None. C.T. Juel: None. K.D. Galsgaard: None. J. Hunt: None. J.F. Rehfeld: None. K. Rigbolt: Employee; Self; Gubra. Stock/Shareholder; Self; Gubra. S.U. Friis: Stock/Shareholder; Self; Alk-Abello B, Bavarian Nordic, H. Lundbeck A/S, Lundbeck, Novo Nordisk A/S. C. Orskov: None. S.S. Poulsen: None. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. T. Vilsbøll: None. F.K. Knop: Advisory Panel; Self; AstraZeneca, MedImmune, Merck Sharp & Dohme Corp., Mundipharma, Novo Nordisk A/S, Sanofi. Consultant; Self; Amgen Inc., Carmot Therapeutics, Novo Nordisk A/S. Research Support; Self; AstraZeneca, Novo Nordisk A/S. Speaker's Bureau; Self; AstraZeneca, MedImmune, Merck Sharp & Dohme Corp., Mundipharma, Norgine, Novo Nordisk A/S. Funding European Foundation for the Study of Diabetes; Novo Nordisk Foundation; Aase og Ejnar Danielsens Foundation; A.P. Møller Foundation

Enhanced glucagon-like peptide-1 (GLP-1) secretion is important for improved glycemic control after Roux-en-Y gastric bypass (RYGB) and higher concentrations of circulating bile acids (BA) have been suggested to be involved. The BA chenodeoxycholic acid (CDCA) stimulates GLP-1 secretion after RYGB in the absence of nutrients. In this study we investigated the effects of oral intake of CDCA or the BA sequestrant colesevelam (COL) on postprandial GLP-1 secretion in RYGB operated subjects. We hypothesized that CDCA would increase secretion and that COL would decrease secretion by reducing re-absorption of endogenous BA. Twelve participants (BMI 31±2 (mean±SEM) kg/m2, age 43±3 years, time from RYGB 4.3±0.5 years, weight loss after RYGB 34±5 kg) were studied in a single-blinded, randomised study with four experimental days: 1) A mixed meal consisting of solid/semisolid components (1523 kJ, 53E% carb, 33E% fat, 14E% prot) 2) Mixed meal added 1250 mg CDCA 3) Mixed meal added COL 3750 mg 4) Mixed meal with COL administered both as 3750 mg tablets the evening before the experiment and as 3750 mg added to the meal. Oral intake of CDCA increased GLP-1 significantly compared with mixed meal alone (p=0.03), whereas neither single (p=0.74) nor double (p=0.28) dosage of COL affected GLP-1 secretion (Incremental AUC (iAUC) GLP-1: Meal: 2792±288 pmol x min, CDCA: 4362±618, COL: 2703±330, COLx2: 3328±380). Plasma glucose and C-peptide (iAUC) decreased after CDCA but both were unchanged after COL administration vs. meal alone. Beta-cell function (AUC C-peptide/AUC plasma glucose) was equal between all study days. In summary, administration of the exogenous BA CDCA potentiated mixed-meal stimulated GLP-1 secretion after RYGB but did not affect beta-cell function. Administration of the BA sequestrant COL did not affect GLP-1 secretion, glucose concentrations or beta-cell function, suggesting a limited role for endogenous BA for regulation of postprandial glucose metabolism after RYGB. Disclosure M.S. Svane: None. I. Jonsson: None. V. Kristiansen: None. R.E. Kuhre: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. S. Madsbad: None. K.N. Bojsen-Moller: None. Funding European Research Council (695069)

We have recently shown a hyperglucagonemic response to meal test and oral glucose tolerance test (OGTT) in totally pancreatectomized (PX) patients. Here, we employed a glucagon receptor antagonist (GRA) to determine the physiological implications of extrapancreatic glucagon secretion in PX patients. In a double-blinded, randomized, cross-over study, 9 PX patients (age: 61.3±4.3 [mean±SEM] years; BMI: 22.6±1.6 kg/m2) and 9 matched healthy controls (age 65.9±2.9 years; BMI: 23.9±0.9 kg/m2) were examined during two 3-hour 75-OGTTs. Before the tests, subjects ingested 300 mg of the GRA, LY2409021 (Eli Lilly and Company), and placebo, respectively. Patients received their regular dose of insulin the night before each experimental day, but no insulin was given during the experiments. In the healthy controls, the GRA resulted in lower fasting levels of glucose (4.3±0.1 vs. 5.2±0.1 mmol/l, P=0.001), but higher post-OGTT plasma glucose excursions (as assessed by baseline-subtracted area under the curve (bsAUC): 926±92 vs. 467±72 mmol/l×min, P=0.002) compared to placebo. In the PX patients, the GRA and placebo resulted in similar fasting plasma glucose concentrations (11.3±0.9 vs. 12.5±0.8 mmol/l, P=0.266) and post-OGTT glucose excursions (bsAUC: 1,671±96 vs. 1,693±152 mmol/l×min, P=0.398), respectively. In the healthy controls, the GRA resulted in significantly higher fasting concentrations of glucagon (33.1±5.5 vs. 13.0±3.7 pmol/l, P=0.023), which, nevertheless, were suppressed by OGTT to similarly low levels as observed with placebo. In the PX patients, equal fasting glucagon concentrations (3.2±0.7 vs. 4.7±1.9 pmol/l, P=0.373) and similarly exaggerated plasma glucagon responses to OGTT were observed with the GRA and placebo (bsAUC: 1,636±573 vs. 1,034±542 pmol/l×min, P=0.113). In this study, antagonizing the glucagon receptor in totally pancreatectomized patients did not influence fasting or post-OGTT glucose levels. Disclosure C.T.B. Juel: None. A.B. Lund: None. J.F. Rehfeld: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. T. Vilsbøll: None. F.K. Knop: Advisory Panel; Self; AstraZeneca, MedImmune, Merck Sharp & Dohme Corp., Mundipharma, Novo Nordisk A/S, Sanofi. Consultant; Self; Amgen Inc., Carmot Therapeutics, Novo Nordisk A/S. Research Support; Self; AstraZeneca, Novo Nordisk A/S. Speaker's Bureau; Self; AstraZeneca, MedImmune, Merck Sharp & Dohme Corp., Mundipharma, Norgine, Novo Nordisk A/S.

Background: Roux-en-Y gastric bypass (RYGB) is a successful treatment for diabetes and obesity, possibly because it augments post-prandial secretion of the gut hormones glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM) and peptide YY (PYY). Subcutaneous infusion of GLP-1, OXM and PYY (GOP) mimicking hormone levels observed after RYGB reduces food intake. We hypothesized that GOP given for 4 weeks to patients with diabetes and obesity would improve glycaemia and body weight in comparison to placebo. Methods: 26 patients were randomized to a single-blinded GOP or Saline infusion. We also studied 21 patients who had RYGB, and 22 patients on a 800 kcal/day very low calorie diet (VLCD). Outcome measures were: (a) fructosamine levels; (b) body weight; (c) glucose and insulin during mixed meal test (MMT); (d) energy expenditure (EE); (e) energy intake (EI); (f) glucose variability. Findings: GOP infusion was well tolerated and led to a significantly greater mean reduction in fructosamine of -44·1 [95% CI 62·7, -25·5] µmol/L vs. Saline -11·7 [-18·9, -4·5] (difference in treatment effect 32·4 [12·9, 51·9], p=0·002). RYGB and VLCD also led to improvements (-34·0 [-45·6, -22.4] and -28·5 [-40.4, -16.7] respectively). The weight loss with GOP was -4·4 [-5·4, -3·5] kg compared to -2·5 [-4·1, -0·9] with Saline, -10·3 [-11·8, -8·8] with RYGB and -8·3 [-9·5, -7·1] with VLCD. Strikingly, there were key differences in glucose and insulin dynamics during the MMT. After RYGB there was an early glucose peak followed by marked insulin secretion, whereas GOP led to a flat euglycaemic response with no sharp insulin peak. The improvement in glucose tolerance with GOP was superior to VLCD. Interpretation: GOP infusion improves glycaemia and reduces body weight, supporting triple agonism of GLP-1, glucagon and peptide YY receptors as a treatment strategy for diabetes with obesity. Disclosure P. Behary: None. G. Tharakan: None. K. Alexiadou: None. N.A. Johnson: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp. J. Cuenco: None. D. Hope: None. W. Dhillo: None. J.S. Minnion: None. G. Frost: Consultant; Self; Heptares, Nestlé. C. le Roux: Advisory Panel; Self; GI Dynamics, Inc., Herbalife International of America, Inc., Janssen Pharmaceuticals, Inc., Johnson & Johnson, Novo Nordisk A/S. Speaker's Bureau; Self; Merck Sharp & Dohme Corp. S. Purkayastha: None. K. Moorthy: None. J.J. Holst: Advisory Panel; Self; Novo Nordisk A/S. A. Ahmed: None. T. Prevost: None. S. Bloom: None. T.M.M. Tan: Other Relationship; Self; Novo Nordisk A/S. Funding Medical Research Council UK; Imperial Biomedical Research Centre