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Context Fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) are increased in type 2 diabetes and are potential regulators of metabolism. The effect of changes in caloric intake and macronutrient composition on their circulating levels in patients with type 2 diabetes are unknown. Objective To explore the effects of a carbohydrate-reduced high-protein diet with and without a clinically significant weight loss on circulating levels of FGF21 and GDF15 in patients with type 2 diabetes. Methods We measured circulating FGF21 and GDF15 in patients with type 2 diabetes who completed 2 previously published diet interventions. Study 1 randomized 28 subjects to an isocaloric diet in a 6 + 6-week crossover trial consisting of, in random order, a carbohydrate-reduced high-protein (CRHP) or a conventional diabetes (CD) diet. Study 2 randomized 72 subjects to a 6-week hypocaloric diet aiming at a ∼6% weight loss induced by either a CRHP or a CD diet. Fasting plasma FGF21 and GDF15 were measured before and after the interventions in a subset of samples (n = 24 in study 1, n = 66 in study 2). Results Plasma levels of FGF21 were reduced by 54% in the isocaloric study (P < .05) and 18% in the hypocaloric study (P < .05) in CRHP-treated individuals only. Circulating GDF15 levels increased by 18% (P < .05) following weight loss in combination with a CRHP diet but only in those treated with metformin. Conclusion The CRHP diet significantly reduced FGF21 in people with type 2 diabetes independent of weight loss, supporting the role of FGF21 as a “nutrient sensor.” Combining metformin treatment with carbohydrate restriction and weight loss may provide additional metabolic improvements due to the rise in circulating GDF15., Context: Fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) are increased in type 2 diabetes and are potential regulators of metabolism. The effect of changes in caloric intake and macronutrient composition on their circulating levels in patients with type 2 diabetes are unknown. Objective: To explore the effects of a carbohydrate-reduced high-protein diet with and without a clinically significant weight loss on circulating levels of FGF21 and GDF15 in patients with type 2 diabetes. Methods: We measured circulating FGF21 and GDF15 in patients with type 2 diabetes who completed 2 previously published diet interventions. Study 1 randomized 28 subjects to an isocaloric diet in a 6 + 6-week crossover trial consisting of, in random order, a carbohydrate-reduced high-protein (CRHP) or a conventional diabetes (CD) diet. Study 2 randomized 72 subjects to a 6-week hypocaloric diet aiming at a ∼6% weight loss induced by either a CRHP or a CD diet. Fasting plasma FGF21 and GDF15 were measured before and after the interventions in a subset of samples (n = 24 in study 1, n = 66 in study 2). Results: Plasma levels of FGF21 were reduced by 54% in the isocaloric study (P < .05) and 18% in the hypocaloric study (P < .05) in CRHP-treated individuals only. Circulating GDF15 levels increased by 18% (P < .05) following weight loss in combination with a CRHP diet but only in those treated with metformin. Conclusion: The CRHP diet significantly reduced FGF21 in people with type 2 diabetes independent of weight loss, supporting the role of FGF21 as a “nutrient sensor.” Combining metformin treatment with carbohydrate restriction and weight loss may provide additional metabolic improvements due to the rise in circulating GDF15.

A physiological feedback system exists between hepatocytes and the alpha cells, termed the liver-alpha cell axis and refers to the relationship between amino acid-stimulated glucagon secretion and glucagon-stimulated amino acid catabolism. Several reports indicate that non-alcoholic fatty liver disease (NAFLD) disrupts the liver-alpha cell axis, because of impaired glucagon receptor signaling (glucagon resistance). However, no experimental test exists to assess glucagon resistance in humans. The objective was to develop an experimental test to determine glucagon sensitivity with respect to amino acid and glucose metabolism in humans. The proposed glucagon sensitivity test (comprising two elements: 1) i.v. injection of 0.2 mg glucagon and 2) infusion of mixed amino acids 331 mg/hour/kg) is based on nine pilot studies which are presented. Calculation of a proposed glucagon sensitivity index with respect to amino acid catabolism is also described. Secondly, we describe a complete study protocol (GLUSENTIC) according to which the glucagon sensitivity test will be applied in a cross-sectional study currently taking place. 65 participants including 20 individuals with a BMI 18.6–25 kg/m2, 30 individuals with a BMI ≥ 25–40 kg/m2, and 15 individuals with type 1 diabetes with a BMI between 18.6 and 40 kg/m2 will be included. Participants will be grouped according to their degree of hepatic steatosis measured by whole-liver magnetic resonance imaging (MRI). The primary outcome measure will be differences in the glucagon sensitivity index between individuals with and without hepatic steatosis. Developing a glucagon sensitivity test and index may provide insight into the physiological and pathophysiological mechanism of glucagon action and glucagon-based therapies.

Background: 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. Methods: 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). Results: 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. Conclusions: 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.

Glucagon and insulin are the main regulators of blood glucose. While the actions of insulin are extensively mapped, less is known about glucagon. Besides glucagon's role in glucose homeostasis, there are additional links between the pancreatic alpha cells and the hepatocytes, often collectively referred to as the liver-alpha cell axis, which may be of importance for health and disease. Thus, glucagon receptor antagonism (pharmacological or genetic), which disrupts the liver-alpha cell axis, not only results in lower fasting glucose, but also in reduced amino acid turnover, and dyslipidemia. Here, we review the actions of glucagon on glucose homeostasis, amino acid catabolism, and lipid metabolism in the context of the liver-alpha cell axis. The concept of glucagon resistance is also discussed, and we argue that the various elements of the liver-alpha cell axis may be differentially affected in metabolic diseases such as diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD). This conceptual rethinking of glucagon biology may explain why patients with type 2 diabetes have hyperglucagonemia and how NAFLD disrupts the liver-alpha cell axis, compromising the normal glucagon-mediated enhancement of substrate-induced amino acid turnover and possibly fatty acid beta-oxidation. Glucagon-induced glucose production may, in contrast to amino acid catabolism, however not be affected by NAFLD explaining the diabetogenic effect of NAFLD-associated hyperglucagonemia. Consideration of the liver-alpha cell axis is essential to understand the complex pathophysiology underlying diabetes and other metabolic diseases.

Context: Inhibitors of the protease neprilysin (NEP) are used for treating heart failure, but are also linked to improvements in metabolism. NEP may cleave proglucagon-derived peptides, including the glucose and amino acid (AA)-regulating hormone glucagon. Studies investigating NEP inhibition on glucagon metabolism are warranted.Objective: This work aims to investigate whether NEP inhibition increases glucagon levels.Methods: Plasma concentrations of glucagon and AAs were measured in eight healthy men during a mixed meal with and without a single dose of the NEP inhibitor/angiotensin II type 1 receptor antagonist, sacubitril/valsartan (194 mg/206 mg). Long-term effects of sacubitril/valsartan (8 weeks) were investigated in individuals with obesity (n = 7). Mass spectrometry was used to investigate NEP-induced glucagon degradation, and the derived glucagon fragments were tested pharmacologically in cells transfected with the glucagon receptor (GCGR). Genetic deletion or pharmacological inhibition of NEP with or without concomitant GCGR antagonism was tested in mice to evaluate effects on AA metabolism.Results: In healthy men, a single dose of sacubitril/valsartan significantly increased postprandial concentrations of glucagon by 228%, concomitantly lowering concentrations of AAs including glucagonotropic AAs. Eight-week sacubitril/valsartan treatment increased fasting glucagon concentrations in individuals with obesity. NEP cleaved glucagon into 5 inactive fragments (in vitro). Pharmacological NEP inhibition protected both exogenous and endogenous glucagon in mice after an AA challenge, while NEP-deficient mice showed elevated fasting and AA-stimulated plasma concentrations of glucagon and urea compared to controls.Conclusion: NEP cleaves glucagon, and inhibitors of NEP result in hyperglucagonemia and may increase postprandial AA catabolism without affecting glycemia.

Glucagon regulates the hepatic amino acid metabolism and increases ureagenesis. Ureagenesis is activated by N-acetylglutamate (NAG), formed via activation of N-acetylglutamate synthase (NAGS). With the aim to identify the steps whereby glucagon both acutely and chronically regulates ureagenesis, we investigated whether glucagon receptor-mediated activation of ureagenesis is required in a situation where NAGS activity and/or NAG levels are sufficient to activate the first step of the urea cycle in vivo. Female C57BL/6JRj mice treated with a glucagon receptor antagonist (GRA), glucagon receptor knockout (Gcgr-/-) mice, and wild-type (Gcgr+/+) littermates received an intraperitoneal injection of N-carbamoyl glutamate (Car; a stable variant of NAG), L-citrulline (Cit), Car and Cit (Car + Cit), or PBS. In separate experiments, Gcgr-/- and Gcgr+/+ mice were administered N-carbamoyl glutamate and L-citrulline (wCar + wCit) in the drinking water for 8 wk. Car, Cit, and Car + Cit significantly (P< 0.05) increased plasma urea concentrations, independently of pharmacological and genetic disruption of glucagon receptor signaling (P = 0.9). Car increased blood glucose concentrations equally in GRA- and vehicle-treated mice (P = 0.9), whereas the increase upon Car + Cit was impaired in GRA-treated mice (P = 0.008). Blood glucose concentrations remained unchanged in Gcgr-/- mice upon Car (P = 0.2) and Car + Cit (P = 0.9). Eight weeks administration of wCar + wCit did not change blood glucose (P > 0.2), plasma amino acid (P > 0.4), and urea concentrations (P > 0.3) or the area of glucagon-positive cells (P > 0.3) in Gcgr-/- and Gcgr+/+ mice. Our data suggest that glucagon-mediated activation of ureagenesis is not required when NAGS activity and/or NAG levels are sufficient to activate the first step of the urea cycle.