Role of melanocortin3 receptor in the dorsomedial nucleus of the hypothalamus food entrainment oscillator

Peiran Yang1, Oliver Marston1, Karima Begriche2, Andrew Butler2, Lora Heisler1. 1Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, United Kingdom, 2The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, United States.

The melanocortin system is a critical component of energy homeostasis and agonists of melanocortin3- and melanocortin4-receptors (MC3R and MC4R) have been a major drug discovery target for the pharmaceutical industry for the treatment of obesity. Agonism of MC4R and MC3R via administration of the mixed MC3/4 agonist melanotan II elicits anorexia, an effect partially attenuated in both Mc3r and Mc4r knockout mice. While the MC3R is required for normal energy homeostasis, how this receptor influences appetitive behaviour is incompletely understood. The recent discovery that functional MC3Rs are required for appropriate expression of food anticipatory behaviour, together with the dense expression of MC3R mRNA within the dorsomedial hypothalamic nucleus (DMH), a structure implicated in food entrainment processes and purported to be one possible site of the ‘food-entrainable oscillator’, led us to hypothesize that MC3R function within this structure may be important in the mediation of entrainment to scheduled food presentation. We tested this hypothesis by assessing changes in c-FOS-immunoreactivity (a marker of neuronal activation) in wild-type and MC3R-knockout mice subjected to either ad libitum or scheduled (restricted food availability) feeding regimes.

In the first study, adult C56BL/6 wild type mice, maintained on a 12 light/dark period were provided with a chow diet either ad libitum or for 4 hrs corresponding to zeitgeber time (5-9) for 1 week. Mice were culled at zeitgeber time 6.5 and assessed for c-FOS expression using immunohistochemistry. Compared to ad libitum fed mice (n = 3), restricted feeding (n = 5) resulted in a highly significant upregulation of c-FOS-immunoreactivity within the DMH (Prostral = 0.0050, Pmedial = 0.0005, Pcaudal = 0.0013). In the second study, we investigated whether MC3R function is important in mediating the response to scheduled feeding observed in study 1. Wild type and Mc3r knockout mice underwent the restricted feeding paradigm. Mc3r knockout mice (n = 7) expressed significantly fewer (Pmedial = 0.0227) c-FOS labelled nuclei in the intermediate level of the DMH compared to wild type controls (n = 9). However, c-FOS-immunoreactivity in the rostral and caudal DMH was not significantly different (Prostral = 0.9927, Pcaudal = 0.3193). Because entrainment to scheduled feeding is reported to occur independently of the master circadian oscillator located in the suprachiasmatic nucleus of the hypothalamus (SCN), we also assessed c-FOS-immunoreactivity in the SCN of the same wild type and Mc3r knockout animals. We found that there was no significant difference in c-FOS expression at any level of the SCN examined (Prostral = 0.7818, Pmedial = 0.9872, Pcaudal = 0.6793).

Here we demonstrate that a restricted feeding regime comprised of scheduled food presentation increases c-FOS expression within the DMH. The attenuated response in Mc3r knockout animals suggests that MC3R-expressing neurons within the intermediate DMH may be involved in the entrainment process. Further, the absence of change in c-FOS immunoreactivity within the SCN supports the hypothesis that scheduled food entrainment is SCN independent. These data suggest that one mechanism through which the MC3Rs modulate energy balance is through the food entrainment oscillator in the DMH.