Elsevier

Neuropeptides

Volume 48, Issue 3, June 2014, Pages 143-151
Neuropeptides

Double deletion of orexigenic neuropeptide Y and dynorphin results in paradoxical obesity in mice

https://doi.org/10.1016/j.npep.2014.03.001Get rights and content

Abstract

Objective

Orexigenic neuropeptide Y (NPY) and dynorphin (DYN) regulate energy homeostasis. Single NPY or dynorphin deletion reduces food intake or increases fat loss. Future developments of obesity therapeutics involve targeting multiple pathways. We hypothesised that NPY and dynorphin regulate energy homeostasis independently, thus double NPY and dynorphin ablation would result in greater weight and/or fat loss than the absence of NPY or dynorphin alone.

Design and methods

We generated single and double NPY and dynorphin knockout mice (NPYΔ, DYNΔ, NPYDYNΔ) and compared body weight, adiposity, feeding behaviour, glucose homeostasis and brown adipose tissue uncoupling protein-1 (UCP-1) expression to wildtype counterparts.

Results

Body weight and adiposity were significantly increased in NPYDYNΔ, but not in NPYΔ or DYNΔ. This was not due to increased food intake or altered UCP-1 expression, which were not significantly altered in double knockouts. NPYDYNΔ mice demonstrated increased body weight loss after a 24-h fast, with no effect on serum glucose levels after glucose injection.

Conclusions

Contrary to the predicted phenotype delineated from single knockouts, double NPY and dynorphin deletion resulted in heavier mice, with increased adiposity, despite no significant changes in food intake or UCP-1 activity. This indicates that combining long-term opioid antagonism with blockade of NPY-ergic systems may not produce anti-obesity effects.

Introduction

The recent decision by the US Food and Drug Authority (FDA) to delay approval of the new anti-obesity drug, Contrave (Orexigen, 2011), has once again reignited the debate of whether the effectiveness of weight-loss drugs outweighs their side effects. Contrave, developed by Orexigen, is a fixed-dose combination of naltrexone, a non-selective opioid receptor antagonist, and bupropion, a selective dopamine reuptake inhibitor (Orexigen, 2011). Due to its cardiovascular effects (Orexigen, 2011), the FDA has sought extensive safety data prior to its approval as an oral obesity treatment. Overweight and obesity currently affect billions of people worldwide, particularly those in developed nations, and are now becoming increasingly prevalent in developing countries also (WHO, 2013). As the rates of this epidemic are rising alarmingly (WHO, 2013), with lifestyle interventions having low success rates (Mann et al., 2007) and surgery being the only effective treatment (Picot et al., 2009), albeit with side effects and not being suitable for everyone, the search for an elusive drug cure is rife.

A large majority of people with a body mass index in the overweight or obese range find that maintaining a reduced body weight in the long-term is a large, compounding hurdle in the quest for a healthy body weight. Up to 70% of those that lose weight using lifestyle interventions alone regain the weight within 4 years (Sumithran and Proietto, 2013, Wing and Phelan, 2005). This is due – at least in part – to weight loss activating adaptive responses that stimulate appetite and reduce energy expenditure, as recently reviewed (Sainsbury and Zhang, 2012). These adaptive responses to energy restriction are seemingly mediated by numerous hypothalamic peptides (Sainsbury and Zhang, 2010, Sainsbury and Zhang, 2012), notably neuropeptide Y (NPY) (Stephens et al., 1995) and possibly also the opioid peptides, dynorphins (Kalra and Kalra, 1996, Sainsbury et al., 2007), with the aforementioned drug, Contrave, utilising opioid receptor antagonism as one of its mechanisms of action.

Negative energy balance, such as during dieting and other lifestyle-based weight loss, leads to decreased circulating leptin levels (Belza et al., 2009) and – at least in rodents – subsequently increased hypothalamic NPY expression (Stephens et al., 1995). Increased central NPY-ergic tonus has been shown to result in increased appetite (Clark et al., 1984) and reduced physical activity (Heilig et al., 1989). These responses are associated with significant decreases in total energy expenditure and body temperature (Hwa et al., 1999), along with increases in food efficiency and fat accretion (Stanley et al., 1986). These effects of NPY contribute to obesity in rodents when leptin action is permanently reduced (Wong et al., 2013), and conceivably also contribute to body weight regain in people after lifestyle-based weight loss interventions (Sainsbury and Zhang, 2010, Sainsbury and Zhang, 2012). Therefore, it would follow on that blocking the effects of increased hypothalamic NPY-ergic tonus, thus blocking these adaptive responses to energy restriction, could increase the effectiveness of weight loss interventions.

In addition to the NPY system, the three peptide families of the endogenous opioid system – endorphins, enkephalins and dynorphins, which preferentially act on mu (μ), delta (δ) and kappa (κ) opioid receptors respectively – are implicated in feeding and body weight regulation (Cooper, 1980, Grandison and Guidotti, 1977, Thornhill et al., 1976). Of these endogenous opioid systems, κ opioid receptors have been particularly implicated in these processes (Arjune and Bodnar, 1990, Hamilton and Bozarth, 1988, Levine et al., 1990). Specific blockade of κ-opioid receptors significantly reduces fasting-induced hyperphagia in rats (Lambert et al., 1993), as well as food intake and body weight in obese rodent models (Cole et al., 1995, Jarosz and Metzger, 2002). Further evidence for a role of κ-opioid receptors in the regulation of energy balance is the observation that dynorphin knockout mice have significantly less white adipose tissue mass and lose more weight during a 24-h fast than wildtype mice (Sainsbury et al., 2007).

Evidence suggests that endogenous opioids may contribute to the energy-conserving, appetite promoting effects of negative energy balance, and that they do so via at least partially distinct and additive pathways to those activated by NPY (Cooper, 1980, Lambert et al., 1993, Sainsbury et al., 2007). NPY and pre-prodynorphin mRNA and protein are co-localised in regions of the hypothalamus involved in energy homeostasis regulation, notably the arcuate nucleus (Lin et al., 2006). After a 24-h fast, there are increases in immunoreactivity levels of both NPY (in arcuate and paraventricular nuclei) and dynorphin (in the overall hypothalamus) (Przewlocki et al., 1983, Sahu et al., 1988), suggesting potentially similar actions. Notably, double intracerebroventricular administration of a NPY antibody and κ-opioid receptor antagonist, norbinaltorphimine, resulted in additive inhibition of hyperphagia, greater than responses seen in single NPY or κ-opioid receptor system disruption (Lambert et al., 1993). This evidence provides support for the possibility that the NPY and dynorphin systems function at least partially independently of one another in the control of energy homeostasis.

Based on these findings of possible independent and hence additive actions of NPY and dynorphin in the regulation of energy balance, and with a view towards future development of pharmacological obesity treatments that target dual pathways, we hypothesised that double ablation of both NPY and dynorphin function would result in greater weight and/or fat loss than the absence of either NPY or dynorphin alone. In this work, we specifically tested this hypothesis using a double NPY and dynorphin knockout mouse model (NPYDYNΔ), with subsequent analysis of body weight, adiposity, food intake, brown adipocyte thermogenesis marker expression, as well as blood glucose response to glucose injection.

Section snippets

Ethics statement and animal care

All research and animal care procedures were approved by the Garvan Institute/St Vincent’s Hospital Animal Ethics Committee and in agreement with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. All mice were group housed, unless otherwise stated, under conditions of controlled temperature (22 °C) and illumination (12 h light–dark cycle, lights on at 7:00 h) with ad libitum access to water and standard chow (8% calories from fat, 21% calories from protein,

Body weight is increased after double NPY and dynorphin ablation

Measuring the body weight of wildtype (WT), NPYΔ, DYNΔ and NPYDYNΔ mice from 5 to 13 weeks of age revealed that both male and female NPYDYNΔ mice, as well as female DYNΔ mice, were significantly heavier than their wildtype counterparts (Fig. 1). Importantly, male but not female NPYDYNΔ mice were already heavier than wildtype mice at 5 weeks of age (Fig. 1).

Increased body weight in NPYDYNΔ mice is due to increased adiposity

At the conclusion of the experiment at 13–14 weeks of age, individual white adipose tissue depots were dissected and weighed to determine the

Discussion

The results from this study demonstrate that the simultaneous ablation of both NPY and dynorphin in mice does not induce greater weight or fat losses than changes induced by single deletion of either NPY or dynorphin. In fact, double deletion of NPY and dynorphin resulted in significantly heavier mice, with corresponding and marked increases in adiposity relative to wildtype controls. This was a surprising finding as it has previously been reported, and corroborated, although not significantly

Author contributions

ADN: data analysis and interpretation, literature search, generation of figures, manuscript preparation; KS: data collection; CS: data interpretation, manuscript preparation; NJL; data collection; DB: data collection; LM: data interpretation, manuscript preparation; EY: data collection, manuscript preparation; RFE: data collection; LZ: data interpretation, manuscript preparation; SL: data interpretation, manuscript preparation; YCS: data analysis and interpretation, manuscript preparation; PAB:

Acknowledgements

We thank the staff of the Garvan Institute Biological Testing Facility for facilitation of these experiments. The expert administrative help of Felicity Forsyth of the Garvan Institute in the preparation and submission of this manuscript is gratefully acknowledged.

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