Homeostastic and non-homeostatic functions of melanocortin-3 receptors in the control of energy balance and metabolism

Abstract

The central nervous melanocortin system is a neural network linking nutrient-sensing systems with hypothalamic, limbic and hindbrain neurons regulating behavior and metabolic homeostasis. Primary melanocortin neurons releasing melanocortin receptor ligands residing in the hypothalamic arcuate nucleus are regulated by nutrient-sensing and metabolic signals. A smaller group of primary neurons releasing melanocortin agonists in the nucleus tractus solitarius in the brainstem are also regulated by signals of metabolic state. Two melanocortin receptors regulate energy homeostasis. Melanocortin-4 receptors regulate satiety and autonomic outputs controlling peripheral metabolism. The functions of melanocortin-3 receptors (MC3R) expressed in hypothalamic and limbic structures are less clear. Here we discuss published data and preliminary observations from our laboratory suggesting that neural MC3R regulate inputs into systems governing the synchronization of rhythms in behavior and metabolism with nutrient intake. Mice subjected to a restricted feeding protocol, where a limited number of calories are presented at a 24h interval, rapidly exhibit bouts of increased wakefulness and activity which anticipate food presentation. The full expression of these responses is dependent on MC3R. Moreover, MC3R knockout mice are unique in exhibiting a dissociation of weight loss from improved glucose homeostasis when subject to a restricted feeding protocol. While mice lacking MC3R fed ad libitum exhibit normal to moderate hyperinsulinemia, when subjected to a restricted protocol they develop hyperglycemia, glucose intolerance, and dyslipidemia. Collectively, our data suggest that the central nervous melanocortin system is a point convergence in the control of energy balance and the expression of rhythms anticipating nutrient intake.

Figure 1

MC3R are required for food anticipatory activity. (A) Actograms of averaged wheel running for WT and Mc3r−/− mice. The lights-on and dark period are indicated by the white and black rectangles; the period of food access is indicated by the grey shading. (B) Wheel running data showing activity of ad libitum fed mice, and during restricted feeding (RF; 60% of normal calories provided at 1300 h / ZT7). In both panels, it is clear that the expression of anticipatory activity is severely attenuated in Mc3r−/− mice. (C, D) Increased wakefulness anticipating food presentation is not observed in Mc3r−/− mice. 24h patterns of wakefulness are shown for WT (C, n=9) and Mc3r−/− (D, n=6) mice during ad libitum (AL, solid circles in both panels) or after 3d of restricted feeding (open circles). RF was associated with increased wakefulness in WT mice in the 2h period preceding food presentation and reduced wakefulness in the lights-off phase. In the lights on phase, Mc3r−/− mice exhibited increased wakefulness restricted to the 1h after food presentation. * p<0.05 AL vs. RF. [Adapted from Sutton et al. J. Neuroscience copyright 2008 with permission from The Society for Neuroscience]

Figure 2

Mc3r−/− mice develop insulin resistance when subjected to the restricted feeding protocol. (A, B) Serum glucose (A) and insulin (B) levels in WT and Mc3r−/− mice are similar with ad libitum feeding (left panel). However Mc3r−/− mice exhibit abnormal glucose levels and altered insulinemia when subjected to restricted feeding (right panel). The altered insulin observed in Mc3r−/− mice exhibits a circadian pattern; hypoinsulinemia is observed at ZT0, while hyperinsulinemia is observed at ZT6 and ZT12. (C) Mc3r−/− mice are glucose intolerant compared to Mc4r−/− and WT mice; all groups were subjected to the restricted feeding for 2 weeks. * p<0.05 vs. WT within ZT [Adapted from Sutton et al. FASEB J. copyright 2010 with permission from the Federation of American Societies for Experimental Biology]

Figure 3

Mc3r signaling is required for normal liver clock activity during RF. (A–C) Double-plotted expression pattern of Bmal1 (A), Reverbα (B), and Per2 (C) in liver of WT and Mc3r−/− mice fed ad libitum or subjected to restricted feeding (RF, indicated by the grey bar). (D,E) Double-plotted expression pattern of Bmal1 (D) and Rev-erbα (E) in liver of mice treated i.c.v. with AgRP82–131 or acsf pair-fed a nonlimiting amount of food (4.5 g) at ZT12 (6:00 PM, onset of lights off; left panels) or subjected to RF (right panels). *p< 0.05 vs. corresponding WT. AU, arbitrary units. [Adapted from Sutton et al. FASEB J. copyright 2010 with permission from the Federation of American Societies for Experimental Biology]

Figure 4

Preliminary results from an investigation of circadian rhythms in activity of WT and Mc3r−/− mice subject to RF in constant dark. (A) Averaged wheel running data for WT and Mc3r−/− mice as 30 min. bins. Each bin being the averaged of 3 d of activity after 2 wk of RF. ZT0 is the time of food presentation (1300 h). (B) Analysis of activity data separated into periods between and during meals. For this analysis, we separated this period into FAA (ZT-2 to ZT0 - the two hours prior to food presentation), the first and second hour after food presentation (most food is consumed in the first hour [81, 107]), and the period of post meal activity (ZT4 to ZT8). ZT+10 to ZT-6 is the 8 h period between meals when WT showed minimal activity. As predicted, WT subject to RF exhibited a period of increased activity of about 12 h (ZT-4 to ZT+8) centered around food presentation. As a group, Mc3r−/− mice appeared to exhibit FAA, with no significant difference from controls. However, activity between meals and in the first hour after food presentation was markedly different: they exhibited more activity the hour following food presentation (*, p<0.05), and were less active after the meal (**, p<0.01). (C-E) Analysis of individual mice indicated that the differences in activity shown in panel B were due to half of the Mc3r−/− mice not entraining to food presentation. Representative actograms showing activity of individual mice during the ad libitum free running period and subsequent RF are shown for a WT mouse (C), and for two Mc3r−/− mice which did not entrain to RF (D, E). Collectively, the analysis of wheel running data obtained in constant dark indicate that approximately half of the Mc3r−/− mice were not able to entrain their rhythm to nutrient intake after 2 wk of restricted feeding.

Figure 5

Arrythmic circadian rhythms of clock gene expression in the brain and liver of Mc3r−/− mice housed in constant dark. Tissues were collected from WT and Mc3r−/− mice at 6h intervals (0600, 1200, 1800 and 2400 h) 4 days after a phase delay in food presentation, and the expression patterns of clock genes assessed in the brain (A) and liver (B) using published methods [81, 83]. The mice were not fed on the day of tissue collection.