Fig 1: Neprilysin (NEP)-deficient mice have increased fasting and amino acid-stimulated glucagon and urea concentrations. Plasma concentrations of A, glucagon and B, corresponding incremental areas under the curve (iAUCs); C, blood glucose concentrations, plasma concentrations of D, insulin; E, body weight; plasma concentrations of F, amino acids; and G, corresponding iAUCs, plasma concentrations of H, urea; and I, corresponding iAUCs; and plasma concentrations of J, ∆ urea in male NEP–/– mice (black circles) and NEP+/+ mice (open circles)-challenged amino acids (equivalent to 1% of body weight) at time 0 minutes (n = 5-6). Data are presented as mean ± SEM and are analyzed by A, C, D, F, H, and J, repeated-measures 2-way analysis of variance, and B, E, G and I, unpaired t tests. –/– indicates NEP–/– mice and +/+ indicates NEP+/+ mice.
Fig 2: Neprilysin (NEP) degrades glucagon into metabolites (Gcg1-21, Gcg1-19, Gcg1-18, Gcg5-18, and Gcg1-13) that do not activate the glucagon receptor (GCGR). Matrix-assisted laser desorption/ionization time of flight identified m/zs in the m/z spectra 1500 to 4500 after recombinant A, human, and B, mouse NEP (10 ng) mixed with glucagon (1 µg) after 15, 30, and 60 minutes’ incubation, and after 60 minutes’ incubation with glucagon, NEP, and the NEP inhibitor sacubitrilat (0.5 nmol, 10% dimethyl sulfoxide) (active form of the prodrug, sacubitril). C, GCGR activation (dose-response curve) for native glucagon (Gcg1-29)-induced 3′,5′-cyclic adenosine 5′-monophosphate (cAMP) accumulation in COS-7 cells transiently transfected with human GCGR. D, Dose-response curves for cAMP accumulation with glucagon-degradation products in COS-7 cells transiently transfected with human GCGR (3 technical replicates).
Fig 3: NPY levels correlate with liver disease in mice and in human hepatic cirrhosis(A) Serum NPY ELISA from control and cirrhotic patients undergoing transjugular intrahepatic portosystemic shunt (TIPS) treatment, n = 7 (TIPS-group, cirrhotic patients) and n = 6 (controls), ∗∗p < 0.02 for cirrhotic patients vs. healthy individuals (absolute values).(B) Serum NPY ELISA in cirrhotic patients shows increased NPY in Child-Pugh class C compared with Child-Pugh class A patients, n = 5 per group, ∗p < 0.05 for Child-Pugh class C vs. Child-Pugh class A cirrhotic patients. Results are expressed as mean ± standard error of the mean (SEM).(C) Serum NPY ELISA from portal vein (PV, hepatic inflow) and hepatic vein (HV, hepatic outflow) in TIPS patients, n = 16 per group, ∗∗p < 0.02 for PV vs. HV.(D) Serum NPY ELISA from hepatic vein (HV) and hepatic artery (HA) in n = 21 per group, n.s. (not significant).(E) NPY levels compared with portal vein and hepatic artery, ∗∗∗p < 0.001.(F) In silico analysis of hepatic mRNA NPY levels compared with TGFB1 expression in human liver tissues.(G) Hepatic Nep and Col1A1 mRNA expression in patients with chronic liver disease; number of XY pairs = 17, r = 0.7472, p = 0.0006.(H and I) Hepatic Nep mRNA expression from bile duct ligation (BDL)-treated and CCl4-treated WT (C57BL/6) mice for 2 and 4 weeks, respectively, ∗∗p < 0.02 for BDL- or CCl4-treated vs. corresponding control mice. Data were normalized to the expression of 18S RNA.(J) Liver homogenates NPY ELISA from WT and Nep−/− mice, control vs. CCl4, ∗p < 0.05.(K) Morphometric analysis of Sirius red staining of livers from WT mice compared with Nep−/− with and without NPYFL (left panel) and WT BDL mice vs. Nep−/− BDL with and without NPYFL (right panel). n = 5 per group, ∗∗p < 0.01, ∗∗∗p < 0.001, and n.s. (non-significant).(L) Liver sections stain with Sirius red. Scale bar: 200 μm.(M) Col1A1 mRNA expression of WT mice comparing non-treated controls vs. animals that were injected with full-length NPY, ∗∗p < 0.01. Col1A1 mRNA expression of Nep−/− mice after profibrotic stimuli (BDL) comparing control vs. Nep−/− after injecting full-length NPY, ∗∗p < 0.01. Data were normalized to the expression of 18S RNA.
Fig 4: Nep deletion increases HSC activation in BDL- and CCl4-treated mice(A and B) Hepatic αSMA IHC in BDL- and CCl4-treated Nep−/− mice and WT mice with their respective morphometric analysis. Scale bar: 200 μm.(C) Hepatic Acta2 mRNA levels in BDL- and CCl4-treated Nep−/− mice and WT mice. All data were normalized to the expression of 18S RNA.(D) In silico analysis of NEP (MME) levels compared with ACTA2 expression in human non-tumorous liver tissue samples.(E) Western blot analysis from controls vs. CCl4-treated Nep−/− mice developed with αSMA antibody. The expression of GAPDH was used as a loading control. Results are expressed as mean ± SEM; n = 5 per group, ∗p < 0.05,∗∗p < 0.01 for BDL- or CCl4-treated vs. corresponding control mice.
Fig 5: Oral administration of Entresto to fibrotic mice improves fibrosis and lowers portal hypertension(A) Diagram of Entresto effects. Dual inhibition of NEP (sacubitril) (red) and AT1R (valsartan) (red) decreases fibrosis and contraction together with portal pressure. Although full-length NPY enhances contraction through activation of the Y1R and Gαi-adenylate cyclase (AD) (green arrows), the blockade of AT1R by valsartan cannot be enhanced by the action of AD, so contraction is decreased.(B) Portal pressure measurements showed a significant reduction in both Entresto-treated mice groups. Results are expressed as mean ± SEM; n = 5 per group, ∗∗p < 0.01 for Entresto-treated mice vs. corresponding control mice.(C) Hepatic Col1a1 mRNA levels in Entresto-treated mice compared with control mice, n = 5/6 per group, ∗∗p < 0.01. All data were normalized to the expression of 18S RNA.(D) Liver sections stained with Sirius red and hepatic αSMA IHC with their respective morphometric analysis. Scale bar: 200 μm.(E) Western blot analysis of Col1a1 and αSMA proteins, in Entresto-treated mice compared with their respective controls. The expression of GAPDH was used as a loading control. n = 3 per group, ∗p < 0.05,∗∗p < 0.01 for Col1a1 or αSMA vs. GAPDH.
Supplier Page from R&D Systems, a Bio-Techne Brand for Neprilysin/CD10 Protein
Available conjugates: Sizes Available: 10 ug