New function for an old enzyme: NEP deficient mice develop late-onset obesity

Abstract

Background: According to the World Health Organization (WHO) there is a pandemic of obesity with approximately 300 million people being obese. Typically, human obesity has a polygenetic causation. Neutral endopeptidase (NEP), also known as neprilysin, is considered to be one of the key enzymes in the metabolism of many active peptide hormones.

Methodology/principal findings: An incidental observation in NEP-deficient mice was a late-onset excessive gain in body weight exclusively from a ubiquitous accumulation of fat tissue. In accord with polygenetic human obesity, mice were characterized by deregulation of lipid metabolism, higher blood glucose levels, with impaired glucose tolerance. The key role of NEP in determining body mass was confirmed by the use of the NEP inhibitor candoxatril in wild-type mice that increased body weight due to increased food intake. This is a peripheral and not a central NEP action on the switch for appetite control, since candoxatril cannot cross the blood-brain barrier. Furthermore, we demonstrated that inhibition of NEP in mice with cachexia delayed rapid body weight loss. Thus, lack in NEP activity, genetically or pharmacologically, leads to a gain in body fat.

Conclusions/significance: In the present study, we have identified NEP to be a crucial player in the development of obesity. NEP-deficient mice start to become obese under a normocaloric diet in an age of 6-7 months and thus are an ideal model for the typical human late-onset obesity. Therefore, the described obesity model is an ideal tool for research on development, molecular mechanisms, diagnosis, and therapy of the pandemic obesity.

Figure 1. Age-related obesity in NEP-deficient mice.

(a) Comparison of a NEP-knockout (NEP −/−) mouse with an age-matched wild-type animal (NEP +/+). (b) Steatosis of thorax and heart in a NEP-knockout mouse. (c) Abdominal fat accumulation in a NEP-deficient animal. Age-dependent development of body weight in (d) females and (e) males. Data is presented as means ± SEM. Where not shown, error bars lie within the dimensions of the symbols. Average per group 22 mice. Genotype effects ***P<0.001 by two-way ANOVA. Significant differences at specific times are calculated by Bonferroni post-hoc test, **P<0.01 and ***P<0.001. (f) Daily food consumption in 7 and >11 month-old female NEP-knockout (NEP −/−) mice compared with age-matched wild-type animal (NEP +/+), *P<0.05 and **P<0.01.

Figure 2. Body composition and diet-depending weight development in NEP-deficient mice.

NMR-monitored influence of feeding on age-dependent body composition in mice fed with low fat diet divided in (a) muscle masses, (b) free body fluid, (c) fat masses, and (d) fat masses per body weight. Effects of (e) low fat and (f) high fat diet on the development of body mass. Data is presented as means ± SEM of at least 10 animals per group. Two-way ANOVA **P<0.01; ***P<0.001. Where not shown, error bars lie within the dimensions of the symbols. Genotype effects are calculated by two-way ANOVA (**P<0.01; ***P<0.001). Significant differences at specific time-points are calculated by Bonferroni post-hoc test, *P<0.05 and **P<0.01.

Figure 3. Biochemical parameters in obese NEP-deficient mice under low fat diet.

(a) Serum triglycerides in one-year old NEP-deficient animals (−/−) and their age-matched wild-type controls (+/+). Student's t-test ***P<0.001 versus wild-type. (b) Serum HDL (left panel) and VLDL (right panel) in both genotypes. Data is presented as means ± SEM. Student's t-test ***P<0.001 versus wild-type. (c) Significant correlation of serum leptin levels in NEP-deficient mice with increasing body weight (r² 0.92 [Pearson correlation]). (d) Comparison of basic glucose values (before glucose tolerance test) in plasma of NEP-knockout mice with wild-type animals. Student's t-test **P<0.01 versus wild-type. (e) Comparison of NEP-knockout mice (dotted line) with wild-type animals (solid line) in their response on a glucose tolerance test. Treatment differences are calculated by two-way ANOVA *P<0.05.

Figure 4. Effects of candoxatril (Pfizer, UK79,300) on body mass development.

(a) Development of body weight in male C57BL/6 mice starting in an age of 6 months fed with standard diet, supplemented with placebo (solid line and squares) or of the NEP inhibitor candoxatril [consumption 200 mg/kg/day] (broken line and triangles). Treatment day 0 is the first day of treatment. Treatment effect ***P<0.001 by two-way ANOVA. Significant differences at specific time-points are calculated by Bonferroni post-hoc test, *P<0.05 and **P<0.01. (b) Abdominal fat in male C57BL/6 mice after feeding with standard food (open column) or with food supplemented with the NEP inhibitor candoxatril (black column) for two months [consumption 200 mg/kg/day]. Student's t-test **P<0.01 versus placebo. (c) Development of food intake in male C57BL/6 mice fed with standard food (open columns) or the same food supplemented with the NEP inhibitor candoxatril (black columns) [consumption 200 mg/kg/day]. Student's t-test **P<0.01, ***P<0.001 versus placebo. (d) Development of NEP activity in kidney (left panel) and brain (right panel) of mice after oral treatment with candoxatril (1) Placebo, (2) 100 mg/kg/day candoxatril, (3) 200 mg/kg/day candoxatril. Student's t-test *P<0.05, **P<0.01 versus placebo. (e) Development of body weight in male tumor-bearing mice (Pancreas carcinoma, PSN-1) fed with standard food (solid line and squares) or standard food supplemented with the NEP inhibitor candoxatril (200 mg/kg/day, broken line and triangles). Treatment effect *P<0.05, by two-way ANOVA. Significant differences at specific time-points are calculated by Bonferroni post-hoc test, *P<0.05 and **P<0.01.