Improved glycaemia in high-fat-fed neprilysin-deficient mice is associated with reduced DPP-4 activity and increased active GLP-1 levels

Abstract

Aim/hypothesis: Neprilysin, a widely expressed peptidase, is upregulated in metabolically altered states such as obesity and type 2 diabetes. Like dipeptidyl peptidase-4 (DPP-4), neprilysin can degrade and inactivate the insulinotropic peptide glucagon-like peptide-1 (GLP-1). Thus, we investigated whether neprilysin deficiency enhances active GLP-1 levels and improves glycaemia in a mouse model of high fat feeding.

Methods: Nep ^+/+ and Nep ^-/- mice were fed a 60% fat diet for 16 weeks, after which active GLP-1 and DPP-4 activity levels were measured, as were glucose, insulin and C-peptide levels during an OGTT. Insulin sensitivity was assessed using an insulin tolerance test.

Results: High-fat-fed Nep ^-/- mice exhibited elevated active GLP-1 levels (5.8 ± 1.1 vs 3.5 ± 0.8 pmol/l, p < 0.05) in association with improved glucose tolerance, insulin sensitivity and beta cell function compared with high-fat-fed Nep ^+/+ mice. In addition, plasma DPP-4 activity was lower in high-fat-fed Nep ^-/- mice (7.4 ± 1.0 vs 10.7 ± 1.3 nmol ml^-1 min^-1, p < 0.05). No difference in insulin:C-peptide ratio was observed between Nep ^-/- and Nep ^+/+ mice, suggesting that improved glycaemia does not result from changes in insulin clearance.

Conclusions/interpretation: Under conditions of increased dietary fat, an improved glycaemic status in neprilysin-deficient mice is associated with elevated active GLP-1 levels, reduced plasma DPP-4 activity and improved beta cell function. Thus, neprilysin inhibition may be a novel treatment strategy for type 2 diabetes.

Keywords: DPP-4; GLP-1; Glucose tolerance; High fat diet; Insulin secretion; Mme; Mouse; Nep; Neprilysin.

Fig. 1

Neprilysin deficiency improves the glycaemic status of high-fat fed mice. (a) Body weight and (b) fed glucose levels over time in Nep^+/+ and Nep^−/− mice fed a low- or high-fat diet for 16 weeks. Plasma (c) glucose and (d) insulin levels during an OGTT, (e) blood glucose levels during an ITT and (f) beta cell function after 16 weeks of low- or high-fat feeding. Sequential blood samples were taken from the same mouse for each measure. Data are means ± SEM. (a, b) n=19–22 per group. (c–f) n=6–18 per group. (a–e) White squares, Nep^+/+/low-fat diet; black squares, Nep^+/+/high-fat diet; white circles, Nep^−/−/low-fat diet; black circles, Nep^−/−/high-fat diet. (f) White bars, low-fat diet; black bars, high-fat diet. *p<0.05 vs Nep^−/−. iAUC, incremental AUC; ins, insulin; glu, glucose

Fig. 2

Neprilysin deficiency reduces DPP-4 activity and enhances active GLP-1 levels in high-fat fed mice. (a) Plasma neprilysin activity levels in Nep^+/+ mice over 16 weeks of low- or high-fat feeding. White squares, Nep^+/+/low-fat diet; black squares, Nep^+/+/high-fat diet. (b, c) Plasma (b) DPP-4 activity and (c) active GLP-1 levels in Nep^+/+ and Nep^−/− mice fed a low- or high-fat diet for 16 weeks. Data are means ± SEM; n=9–17. *p<0.05 vs Nep^{+/+, †}p<0.05 vs the low-fat diet. White bars, low-fat diet; black bars, high-fat diet. MNA, methoxy-2-naphthylamine

Fig. 3

Ex vivo pharmacological inhibition of neprilysin activity reduces DPP-4 activity in plasma from high fat fed mice. (a) Neprilysin and (b) DPP-4 activity levels in plasma treated ex vivo with dl-thiorphan or vehicle from Nep^+/+ mice fed a low- or high-fat diet for 16 weeks. (c) DPP-4 activity following in vitro incubation of recombinant neprilysin and DPP-4 with dl-thiorphan. Neprilysin:DPP-4 molar ratios of 1:5 and 1:10 were tested. Incubation of recombinant neprilysin and DPP-4 with diprotin A served as a positive control. Data are means ± SEM. (a, b) n=5 per group. n=3 per Neprilysin:DPP-4 molar ratio. White bars, low-fat diet; black bars, highfat diet. (c) Black bars, 1:5 Neprilysin:DPP-4 molar ratio; White bars, 1:10 Neprilysin:DPP-4 molar ratio. *p<0.05 vs vehicle, ^†p<0.05 vs low-fat diet. MNA, methoxy-2-naphthylamine