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

Abstract

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 To investigate whether NEP inhibition increases glucagon levels. Subjects and 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 (eight 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 weeks sacubitril/valsartan treatment increased fasting glucagon concentrations in individuals with obesity. NEP cleaved glucagon into five 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.

Acute effects on glucose tolerance by neprilysin inhibition in patients with type 2 diabetes

“…We have previously showed that neprilysin cleaves glucagon both in vivo in healthy individuals, and in in vitro experiments. 13,19 While considering the powerful effects of glucagon on hepatic glycogenolysis and gluconeogenesis, it seems probable that the hyperglucagonaemia found after neprilysin inhibition may explain the impairment in glucose tolerance of patients with type 2 diabetes in our study. The effect on glucose concentrations by sacubitril/valsartan could in theory also be explained by increased activity of the sympathetic nervous system.…”

“…Neprilysin activity was measured using a previously described in-house assay. 13 Insulin, C-peptide, fatty acids and triglycerides were analysed using Roche COBAS 8000 system. Clot activator tubes were left at room temperature to coagulate for 30 min, whereas EDTA-tubes were placed on ice immediately until centrifuged at 4 C (2000 g for 10 min).…”

“…In vitro cleavage activity was assayed as reported previously 13 by adding 20 ng of neprilysin (R&D Systems, Abingdon, NB) to 2 μg of atrial natriuretic peptide (ANP; SynPeptide, Shanghai, China) or C-peptide (Schafer-N, Copenhagen, Denmark) in a total volume of 20 μl. Reactions were performed in 50 mM Tris, 0.05% Brij-35, pH 9 and incubated at 37 C. Product development was monitored after 15 min and 60 min by MALDI-TOF analysis.…”

Insulinotropic Effects of Neprilysin and/or Angiotensin Receptor Inhibition in Mice

“…Further, heart failure is associated with insulin resistance ( 66 ) and increased sympathetic nerve activity ( 67 ), the latter known to inhibit insulin secretion and stimulate both hepatic gluconeogenesis and glycogenolysis ( 68 ). Interestingly, treatment of obese hypertensive patients with sacubitril/valsartan for 8 weeks was associated with decreased levels of epinephrine and improved insulin sensitivity in one study ( 4 ), but no effect on fasting glucose levels in a more recent study ( 69 ). To better clarify the role of heart failure in the glycemic effects of sacubitril/valsartan and its components, further studies in a mouse model that recapitulates the clinical features of heart failure in the setting of diabetes would be valuable.…”

Angiotensin Receptor-Neprilysin Inhibitor (ARNI) and Cardiac Arrhythmias

“…Sacubitril/valsartan also significantly lowered plasma triglycerides in HFpEF patients, especially in those with higher baseline triglycerides, while modestly increasing low-and high-density lipoprotein cholesterols [33]. Moreover, one-time sacubitril/valsartan elevated the post-prandial glucagon in healthy individuals, while over 8 weeks, the drug increased fasting glucagon in obese hypertensive individuals, without changes in fasting glucose or amino acids [34], suggesting a potential role of ARNI in modulating dyslipidemia and metabolic diseases (Figure 3).…”