The QRFP neuropeptides have been intensively studied during the past twenty years mostly in regard to their regulation of metabolic functions [28]. Even though most of the findings available have been gained from rodents, it is tempting to generalize the data to other species, including humans, due to highly conserved structure of the peptide and similar receptors distribution [29]. Its orexigenic effect was observed in genetically modified mice, as well as in wild type animals following i.c.v. [9, 10] or direct microinjections into the parenchyma of the medial hypothalamic nuclei [15]. Nevertheless, present observations suggest that administration of QRFP-26 into the LHA leads to anorexigenic effect. Both doses (100 ng and 200 ng) rapidly and effectively decreased animals’ food consumption (Fig. 2), but the lower dose had a stronger and more elongated action (from 5th to 50th min). Our data represent the cumulative value of food intake, therefore the difference between control and treated groups remained significant, besides the change in food consumption occurred within the first 5–10 minutes. The effective doses of QRFP delivered into the brain parenchyma have been established based on our previous experiments [15]. It is important to emphasize that our data were observed on healthy, young, male rodents. On one side, this way we minimized potential confounding factors. At the same time, these restrictions mean limitations for the interpretation of the results, which cannot be directly applicable to the general population.
According to the theory of hypothalamic “feeding and satiety centers”, stimulation of the LH should induce hunger and lead to hyperphagia. However, our findings suggest that QRFP administration induces opposite effects. The question of how that could be possible may arise. According to our hypothesis, involvement of NPY system plays a crucial role in appetite-modifying aspects of QRFP. Indeed, pretreatment with Y1R antagonist BIBP3226 fully prevented the anorexigenic effect caused by the neuropeptide during all the observed period (Fig. 3). Our data, in contrast with previous findings, suggest that laterohypothalamic QRFP may exert inhibiting action on the hypothalamic feeding regulation. QRFP receptors were not detected at the Arc POMC neurons [13], thus assumingly QRFP signaling from the LHA suppresses activity of the NPY neurons themselves. Declined stimulation from the Arc to the LHA hunger center, enriched with Y1R, leptin, and insulin receptors [30] results in lower food consumption. This idea is supported by a recent study in Qrfp-/- mice [31]. Immunofluorescent labelling showed that QRFP neurons comprise a distinct (from orexin and MCH) population of cells in the LHA, densely surrounded by NPY and AgRP immunoreactive terminals. Moreover, αMSH-positive fibers have been localized in the same subregion. Even though αMSH terminals had no clear accumulation towards QRFP neurons, the cells were shown to express melanocortin receptors (MC4), thereby this pathway of regulation cannot be excluded.
However, there might be more than the only one mechanism of negative appetite regulation. For example, QRFP receptors have been detected in the parvocellular part of the paraventricular nucleus (PVN), the area densely populated with corticotropin-releasing factor (CRF) neurons [32]. Stimulation of CRF-gene expression by QRFP external administration or signaling from the LHA could lead to decreased appetite and food intake, as it was proven previously for cousin PrRP neuropeptide [33].
Specific QRFP receptors were shown to share nearly 50% of the amino acidic identity with other G protein-coupled receptor subtypes: NPFF1 and FF2, Orexin R1 and R2, the binding sites of the neuropeptides strongly involved in feeding regulation [34–36]. Consequently, an alternative explanation for the observed effects could be a cross-reactivity of the neuropeptide QRFP with NPFF and orexin systems. Nevertheless, orexin (OX) A study on OX knock-out mice suggests that QRFP-induced food consumption is independent of the OX signaling pathway, since knock-out animals presented the same response on QRFP treatment as wild type [10]. Thus, this mechanism of action is unlikely to be involved. Neuropeptide NPFF system is another possible candidate for mediation of feeding regulation. FF binding sites are widely present across the hypothalamus [37]. Moreover, the general anorexigenic effect of NPFF was proven to act via central µ and ƙ opioid receptors with the mediation of NPY and β-endorphin [38]. These data are supported by other findings: concentration of ƙ receptors was higher, while µ was lower in obese mice compared to lean ones [39]. Other pieces of research suggest the possibility of the opioid system’s role in NPY – induced regulation of feeding (for review see [40]).
Generally, the decrease in food intake may be caused by adverse „side effects” of the applied drugs, e.g. reduced locomotion or developed anxiety and corresponding freezing reaction. Indeed, QRFP knockout mice revealed anxiety-like behavior [31]. In agreement with these data, another study established anxiolytic effect of GPR103 (QRFP) receptor agonist in mice [41]. Interestingly, a recent human study on aggressive individuals revealed that the plasma levels of QRFP positively correlated with the anxiety scores in all the studied subjects and controls [42]. Nevertheless, in our study QRFP applied into the LHA did not modify general locomotion, nor the anxiety level of tested animals (Figs. 4 and 5). Assumingly, the applied doses were small enough to reach the anxiety threshold.
Thus, observes decrease in food intake seems to be linked to appetitive mechanisms. These are promising findings in terms of possible clinical application of QRFP for the treatment of feeding disorders, suggesting that therapeutic doses of the neuropeptide do not cause unwanted side effects.
Additionally, administration of Y1 Ant itself led to enhanced rearing and grooming, and avoidance of the open space (opened arms in EPM) in rats, referring to anxiogenic effect of the drug (Figs. 4 and 5). An important role of NPY system in regulation of anxiety and depression has been reported long ago [43, 44]. In recent studies a significant decrease in NPY mRNA and upregulation of Y1R and Y2R mRNA, along with a significant decrease in expression of the NPY protein in the brains of suicidal subjects was reported [45, 46]. Therefore, our findings support the concept of crucial role of NPY, and particularly Y1 receptor subtype, in stress and anxiety responses [47].
To summarize, the administration of neuropeptide QRFP-26 into the lateral hypothalamic area leads to decrease in food consumption. The anorexigenic effect is most likely to be linked to QRFP-NPY - regulating pathway, as Y1R antagonist BIBP3226 prevented the appetite-modulating effect of QRFP-26. Possible involvement of the CRH, NPFF, opioid, and other neurotransmitter systems may be a subject for further research. QRFP-26 in the applied doses did not modify general locomotion nor anxiety level. At the same time, our observations suggest that Y1 Ant treatment itself induces anxiogenic behavior following the intrahypothalamic microinjections, thus supporting the hypothesis regarding involvement of NPY in stress and anxiety regulation.