Arginine-induced glucagon secretion and glucagon-induced enhancement of amino acid catabolism are not influenced by ambient glucose levels in mice.

Amino acids stimulate the secretion of glucagon, and glucagon receptor signaling regulates amino acid catabolism via ureagenesis, together constituting the liver-α cell axis. Impairment of the liver-α cell axis is observed in metabolic diseases such as diabetes. It is, however, unknown whether glucose affects the liver-α cell axis. We investigated the role of glucose on the liver-α cell axis in vivo and ex vivo. The isolated perfused mouse pancreas was used to evaluate the direct effect of low (3.5 mmol/L) and high (15 mmol/L) glucose levels on amino acid (10 mmol/L arginine)-induced glucagon secretion. High glucose levels alone lowered glucagon secretion, but the amino acid-induced glucagon responses were similar in high and low glucose conditions (P = 0.38). The direct effect of glucose on glucagon and amino acid-induced ureagenesis was assessed using isolated perfused mouse livers stimulated with a mixture of amino acids (VaminR, 10 mmol/L) and glucagon (10 nmol/L) during high and low glucose conditions. Urea production increased robustly but was independent of glucose levels (P = 0.95). To investigate the whole body effects of glucose on the liver-α cell axis, four groups of mice received intraperitoneal injections of glucose-Vamin (2 g/kg, + 3.5 µmol/g, respectively, G/V), saline-Vamin (S/V), glucose-saline (G/S), or saline-saline (S/S). Blood glucose did not differ significantly between G/S and G/V groups. Levels of glucagon and amino acids were similar in the G/V and S/V groups (P = 0.28). Amino acids may overrule the inhibitory effect of glucose on glucagon secretion and the liver-α cell axis may operate independently of glucose in mice.NEW & NOTEWORTHY Glucagon is an essential regulator of our metabolism. Recent evidence suggests that the physiological actions of glucagon reside in amino acid catabolism in the so-called liver-α cell axis, in which amino acids stimulate glucagon secretion and glucagon enhances hepatic amino acid catabolism. Here, it is demonstrated that this feedback system is independent of glycemia possibly explaining why hyperglycemia in diabetes may not suppress α cell secretion.

Insulin-induced membrane permeability to glucose in human muscles at rest and following exercise.

KEY POINTS: Increased insulin action is an important component of the health benefits of exercise, but its regulation is complex and not fully elucidated. Previous studies of insulin-stimulated GLUT4 translocation to the skeletal muscle membrane found insufficient increases to explain the increases in glucose uptake. By determination of leg glucose uptake and interstitial muscle glucose concentration, insulin-induced muscle membrane permeability to glucose was calculated 4 h after one-legged knee-extensor exercise during a submaximal euglycaemic-hyperinsulinaemic clamp. It was found that during submaximal insulin stimulation, muscle membrane permeability to glucose in humans increases twice as much in previously exercised vs. rested muscle and outstrips the supply of glucose, which then becomes limiting for glucose uptake. This methodology can now be employed to determine muscle membrane permeability to glucose in people with diabetes, who have reduced insulin action, and in principle can also be used to determine membrane permeability to other substrates or metabolites. ABSTRACT: Increased insulin action is an important component of the health benefits of exercise, but the regulation of insulin action in vivo is complex and not fully elucidated. Previously determined increases in skeletal muscle insulin-stimulated GLUT4 translocation are inconsistent and mostly cannot explain the increases in insulin action in humans. Here we used leg glucose uptake (LGU) and interstitial muscle glucose concentration to calculate insulin-induced muscle membrane permeability to glucose, a variable not previously possible to quantify in humans. Muscle membrane permeability to glucose, measured 4 h after one-legged knee-extensor exercise, increased ∼17-fold during a submaximal euglycaemic-hyperinsulinaemic clamp in rested muscle (R) and ∼36-fold in exercised muscle (EX). Femoral arterial infusion of NG -monomethyl l-arginine acetate or ATP decreased and increased, respectively, leg blood flow (LBF) in both legs but did not affect membrane glucose permeability. Decreasing LBF reduced interstitial glucose concentrations to ∼2 mM in the exercised but only to ∼3.5 mM in non-exercised muscle and abrogated the augmented effect of insulin on LGU in the EX leg. Increasing LBF by ATP infusion increased LGU in both legs with uptake higher in the EX leg. We conclude that it is possible to measure functional muscle membrane permeability to glucose in humans and it increases twice as much in exercised vs. rested muscle during submaximal insulin stimulation. We also show that muscle perfusion is an important regulator of muscle glucose uptake when membrane permeability to glucose is high and we show that the capillary wall can be a significant barrier for glucose transport.

Glucagon receptor antagonism impairs and glucagon receptor agonism enhances triglycerides metabolism in mice.

OBJECTIVE: Treatment with glucagon receptor antagonists (GRAs) reduces blood glucose but causes dyslipidemia and accumulation of fat in the liver. We investigated the acute and chronic effects of glucagon on lipid metabolism in mice. METHODS: Chronic effects of glucagon receptor signaling on lipid metabolism were studied using oral lipid tolerance tests (OLTTs) in overnight fasted glucagon receptor knockout (Gcgr-/-) mice, and in C57Bl/6JRj mice treated with a glucagon receptor antibody (GCGR Ab) or a long-acting glucagon analogue (GCGA) for eight weeks. Following treatment, liver tissue was harvested for RNA-sequencing and triglyceride measurements. Acute effects were studied in C57Bl/6JRj mice treated with a GRA or GCGA 1 h or immediately before OLTTs, respectively. Direct effects of glucagon on hepatic lipolysis were studied using isolated perfused mouse liver preparations. To investigate potential effects of GCGA and GRA on gastric emptying, paracetamol was, in separate experiments, administered immediately before OLTTs. RESULTS: Plasma triglyceride concentrations increased 2-fold in Gcgr-/- mice compared to their wild-type littermates during the OLTT (P = 0.001). Chronic treatment with GCGR Ab increased, whereas GCGA treatment decreased, plasma triglyceride concentrations during OLTTs (P < 0.05). Genes involved in lipid metabolism were upregulated upon GCGR Ab treatment while GCGA treatment had opposite effects. Acute GRA and GCGA treatment, respectively, increased (P = 0.02) and decreased (P = 0.003) plasma triglyceride concentrations during OLTTs. Glucagon stimulated hepatic lipolysis, evident by an increase in free fatty acid concentrations in the effluent from perfused mouse livers. In line with this, GCGR Ab treatment increased, while GCGA treatment decreased, liver triglyceride concentrations. The effects of glucagon appeared independent of changes in gastric emptying of paracetamol. CONCLUSIONS: Glucagon receptor signaling regulates triglyceride metabolism, both chronically and acutely, in mice. These data expand glucagon´s biological role and implicate that intact glucagon signaling is important for lipid metabolism. Glucagon agonism may have beneficial effects on hepatic and peripheral triglyceride metabolism.

Neprilysin Inhibition Increases Glucagon Levels in Humans and Mice With Potential Effects on Amino Acid Metabolism.

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.