The neuropeptide Y (NPY) Y4 receptor, together with Y1, Y2, Y5 and in some species y6, constitute a group of G-protein coupled receptors that mediate the effects of the so call ‘PP-fold’ peptide family (Blomqvist and Herzog, 1997; Michel et al., 1998). This family consist of NPY, polypeptide YY (PYY) and pancreatic polypeptide (PP) and their biological functions include major control mechanisms that regulate energy homeostasis, reproduction, emotion, and anxiety (Zhang et al., 2011; Tasan et al., 2016; Ip et al., 2019). Recently, human genetic analysis revealed a complex gender-dimorphic relationship between NPY4R and body weight (Aerts et al., 2016; Shebanits et al., 2018). In addition, NPY4R genetic variants are found in obese subjects and subjects with anorexia nervosa (Aerts et al., 2016; Ceccarini et al., 2022), further indicating an involvement of NPY Y4 receptor (NPY-Y4R) signalling in the regulation of energy balance and feeding behaviours. In keeping with findings in human, studies using mutant mouse models also suggested a role of NPY-Y4R signalling specifically in interaction with NPY-Y2R signalling in the control of body weight and energy homeostasis (Sainsbury et al., 2003; Sainsbury et al., 2006; Lee et al., 2008). Thus, mice with dual deletions of Npy4r and Npy2r display a lean phenotype as well as an ameliorated diet-induced or leptin receptor deletion-induced obesity phenotype that is not seen in mice with Npy4r or Npy2r single deletion (Sainsbury et al., 2003, Sainsbury et al., 2006, Lee et al., 2008).

While considerable evidence exists for the involvement of NPY-Y4R in the regulation of energy homeostasis, the underlying mechanisms are less clear. Most our understanding of the role of NPY-4R in the regulation of energy homeostasis came from studies investigating the role of PP. While the NPY-Y4R can be activated by all 3 ligands of the NPY family, pharmacology has revealed that PP has the highest affinity for the NPY-4R and thus been considered as a PP-preferring receptor (Bard et al., 1995; Lundell et al., 1995). PP is a peripheral hormone released from F-type cells of the pancreas in response to a meal or hypoglycaemia (Schwartz, 1983; Berglund et al., 2003). Circulating PP acts as a feed forward inhibitor upon digestive processes thereby influencing food intake and glucose metabolism (Hazelwood, 1993; Asakawa et al., 1999). In line with the peripheral nature of PP, the NPY-4R can be found both in the periphery as well as in parts of the brain that is readily accessible by circulating factors such as the area postrema in the brain stem (Bard et al., 1995; Lundell et al., 1995; Larsen and Kristensen, 1997; Tasan et al., 2009). Importantly however, the NPY-4R is also expressed in brain regions outside the circumventricular organs (for instance the amygdala) (Parker and Herzog, 1999; Fetissov et al., 2004; Tasan et al., 2009), indicating that central NPY-4R signalling, predominantly via NPY stimulation, may mediate effects additional to those of circulating PP and influence other aspects of energy homeostatic control. Indeed, mice lacking Npy4r show an anxiolytic-like phenotype as well as a reduced depression-like behaviours (Painsipp et al., 2008; Tasan et al., 2009), suggesting an involvement of central NPY-4R signalling in the control of emotion that may modulate feeding behaviour (Ip et al., 2019; Ip et al., 2023). In addition, mice lacking Npy4r showed an increased locomotor activity in a novel, but not in the familiar home-cage environment (Painsipp et al., 2008; Tasan et al., 2009; Zhang et al., 2010), indicating that NPY-4R signalling influences physical activity in a context-dependent manner.

In this study, we set out to explore the specific role of NPY-4R signalling in the coordinated regulation of the feeding behaviour and physical activity and how this may influence overall energy homeostasis. Specifically, we investigated the impact of lack of Npy4r on energy intake, feeding behaviours, energy metabolism and physical activity in mice fed on either on standard chow or a high-fat diet. Furthermore, we examined physical activity by both locomotion as well as wheel-running activity, since wheel-running activity has been suggested to engage specific factors in addition to the general tendency of rodents to be physically active and therefore may contribute additional mechanisms that independently alter energy balance and feeding behaviours (Novak et al., 2012).