Gene expression and activity of specific opioid-degrading enzymes in different brain regions of the AA and ANA lines of rats

AbstractThere is increasing evidence that alcoholism runs in families suggesting that genetic factors may play a role. In support of this hypothesis, the alcohol-preferring (AA) and the alcohol-avoiding (ANA) rat lines have been developed through selective outbreeding. Numerous studies indicate that the endogenous opioid system may be involved in controlling ethanol consumption. Changes in opioid peptides and opioid receptors have been described after ethanol intake. But, the influence of ethanol on peptidolytic degradation of opioid peptides has been largely ignored, although the peptidase-mediated metabolism of neuropeptides is known as an important regulatory site of peptidergic transmission. Neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE) degrade neuropeptides, including enkephalin and are expressed in the brain. Furthermore, a good correspondence between the regional distribution of NEP and opioid receptors in rat brain has already been reported pointing to a possible role of NEP in regulating opioid peptides. For both enzymes studied, the gene expression pattern was found to be in good agreement with the corresponding enzyme activities in the brain regions investigated, showing the highest levels for both specific mRNAs and enzyme activities in the striatum. Differences in both measured parameters were detected in distinct brain regions of AA and ANA rats. Furthermore, in some brain regions discrepancies between ACE and NEP mRNA levels and the corresponding enzyme activities were observed. For example, in olfactory bulb and striatum such discrepancies were found for both enzymes studied. In tegmentum/colliculi a higher NEP gene expression in AA rats was associated with a higher NEP enzyme activity compared to the amounts found in ANA rats

New Function for an Old Enzyme: NEP Deficient Mice Develop Late-Onset Obesity

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

The virtually mature BNP (BNP1-32) is a precursor for the more effective BNP1-30

Background and Purpose: The B‐type natriuretic peptide (BNP1‐32) exerts vasorelaxing and cardioprotective activity. BNP is used as a biomarker for the diagnosis of cardiopathological conditions and recombinant BNP1‐32 as a drug for the treatment of such. BNP1‐32 has a short half‐life time and thus, similar to other vasoactive peptides like angiotensin II and bradykinin, can be enzymatically truncated forming bioactive metabolites. We aimed to investigate the metabolism of BNP1‐32 in mouse lung, to identify potential new BNP metabolites and to disclose their biological activity compared to the BNP1‐32, in vitro and in vivo. Experimental Approach: Using High Performance Liquid Chromatography and Mass‐Spectrometry, we identified a new BNP metabolite, BNP1‐30, in the lung being generated by endothelin‐converting enzyme‐1. Key Results: BNP1‐30 is more efficient in stimulating the guanylyl cyclase receptor A (GC‐A) and, in contrast to BNP1‐32, is also able to profoundly stimulate the GC‐B. In vivo, BNP1‐30 reduced the mean arterial blood pressure of normotensive mice after acute infusion significantly more than BNP1‐32. In a model of severe hypertension, a 3‐day infusion of BNP1‐30 was able to reduce systolic blood pressure by 30 mmHg and to improve markers of heart failure, while BNP1‐32 was without significant effect. Conclusion and Implications: Our results suggest that BNP1‐32 is the precursor for the biologically more active BNP1‐30 leading to a fundamental extension of the natriuretic‐peptide system. Due to expanded activity, BNP1‐30 might be a promising treatment option for cardiovascular diseases. Furthermore, its potency as a new diagnostic marker of specific cardiac diseases should be evaluated

Improved Learning and Memory in Aged Mice Deficient in Amyloid β-Degrading Neutral Endopeptidase

BACKGROUND: Neutral endopeptidase, also known as neprilysin and abbreviated NEP, is considered to be one of the key enzymes in initial human amyloid-beta (Abeta) degradation. The aim of our study was to explore the impact of NEP deficiency on the initial development of dementia-like symptoms in mice. METHODOLOGY/PRINCIPAL FINDINGS: We found that while endogenous Abeta concentrations were elevated in the brains of NEP-knockout mice at all investigated age groups, immunohistochemical analysis using monoclonal antibodies did not detect any Abeta deposits even in old NEP knockout mice. Surprisingly, tests of learning and memory revealed that the ability to learn was not reduced in old NEP-deficient mice but instead had significantly improved, and sustained learning and memory in the aged mice was congruent with improved long-term potentiation (LTP) in brain slices of the hippocampus and lateral amygdala. Our data suggests a beneficial effect of pharmacological inhibition of cerebral NEP on learning and memory in mice due to the accumulation of peptides other than Abeta degradable by NEP. By conducting degradation studies and peptide measurements in the brain of both genotypes, we identified two neuropeptide candidates, glucagon-like peptide 1 and galanin, as first potential candidates to be involved in the improved learning in aged NEP-deficient mice. CONCLUSIONS/SIGNIFICANCE: Thus, the existence of peptides targeted by NEP that improve learning and memory in older individuals may represent a promising avenue for the treatment of neurodegenerative diseases

Genetic Deficiency in Neprilysin or Its Pharmacological Inhibition Initiate Excessive Stress-Induced Alcohol Consumption in Mice

Both acquired and inherited genetic factors contribute to excessive alcohol consumption and the corresponding development of addiction. Here we show that the genetic deficiency in neprilysin [NEP] did not change the kinetics of alcohol degradation but led to an increase in alcohol intake in mice in a 2-bottle-free-choice paradigm after one single stress stimulus (intruder). A repetition of such stress led to an irreversible elevated alcohol consumption. This phenomenon could be also observed in wild-type mice receiving an orally active NEP inhibitor. We therefore elucidated the stress behavior in NEP-deficient mice. In an Elevated Plus Maze, NEP knockouts crossed more often the area between the arms, implicating a significant stronger stress response. Furthermore, such animals showed a decreased locomotor activity under intense light in a locomotor activity test, identifying such mice to be more responsive in aversive situations than their wild-type controls. Since the reduction in NEP activity itself does not lead to significant signs of an altered alcohol preference in mice but requires an environmental stimulus, our findings build a bridge between stress components and genetic factors in the development of alcoholism. Therefore, targeting NEP activity might be a very attractive approach for the treatment of alcohol abuse in a society with increasing social and financial stress

Prognostic Significance of Circulating Levels of Hepatocyte Growth Factor in Patients with Chagas’ Disease and Idiopathic Dilated Cardiomyopathy

Objectives: Hepatocyte growth factor (HGF) plays an important role in the improvement in cardiac function and remodeling in a variety of cardiovascular diseases. It is also a strong predictor of mortality in some heart failure (HF) patients. However, its prognostic value in patients with Chagas’ disease (CD) or idiopathic dilated cardiomyopathy (DCM) remains to be investigated. Methods and Results: In this prospective cohort study, HGF concentrations were measured in patients with CD (n = 91), DCM (n = 47), and control subjects (n = 25). While no difference was detected for patients with New York Heart Association class I–II, HGF was significantly increased in advanced HF patients (New York Heart Association class III–IV) in both CD and DCM groups, compared with healthy subjects. There was a strong correlation between HGF and left ventricular ejection fraction in CD patients. However, there was no correlation in patients with DCM. Despite its strong correlation with left ventricular ejection fraction in CD patients, HGF failed to predict mortality and necessity for heart transplant in both CD and DCM patients. Conclusions: Although HGF can be significantly increased in advanced HF patients with CD and DCM, its prognostic value for endpoints is minor. Therefore, the formerly described predictive power for HGF in HF might be restricted to specific etiologies of HF.Peer Reviewe

High-Protein Diet in Lactation Leads to a Sudden Infant Death-Like Syndrome in Mice

BACKGROUND: It is well accepted that reduced foetal growth and development resulting from maternal malnutrition are associated with a number of chronic conditions in later life. On the other hand such generation-transcending effects of over-nutrition and of high-protein consumption in pregnancy and lactation, a proven fact in all developed societies, are widely unknown. Thus, we intended to describe the generation-transcending effects of a high-protein diet, covering most relevant topics of human life like embryonic mortality, infant death, and physical health in later life. METHODS: Female mice received control food (21% protein) or were fed a high protein diet (42% protein) during mating. After fertilisation, females stayed on their respective diet until weaning. At birth, pups were put to foster mothers who were fed with standard food or with HP diet. After weaning, control diet was fed to all mice. All offspring were monitored up to 360 days after birth. We determined glucose-tolerance and measured cardiovascular parameters using a tip-catheter. Finally, abdominal fat amount was measured. RESULTS AND CONCLUSIONS: We identified a worried impact of high-protein diet during pregnancy on dams' body weight gain, body weight of newborns, number of offspring, and also survival in later life. Even more important is the discovery that high-protein diet during lactation caused a more than eight-fold increase in offspring mortality. The observed higher newborn mortality during lactation is a hitherto non-described, unique link to the still incompletely understood human sudden infant death syndrome (SIDS). Thus, although offspring of lactating mothers on high-protein diet might have the advantage of lower abdominal fat within the second half of life, this benefit seems not to compensate the immense risk of an early sudden death during lactation. Our data may implicate that both pregnant women and lactating mothers should not follow classica