Forebrain melanocortin signaling enhances the hindbrain satiety response to CCK-8

Abstract

Melanocortin 4 receptors (MC4R) are hypothesized to mediate the central nervous system actions of leptin to enhance the satiety effects of cholecystokinin (CCK). To further elucidate this mechanism, we confirmed that peripheral administration of CCK-8 is less effective in producing this effect in MC4R-deficient mice (MC4R(-/-)). Whereas intraperitoneal (ip) CCK-8 at 0.75 nmol/kg lean body mass (lbm) suppressed food intake in wild-type mice, CCK-8 doses of 7.5 nmol/kg lbm were required to attenuate food intake in MC4R(-/-) mice. To determine whether melanocortin signaling in the hypothalamic paraventricular nucleus (PVN) participates in regulating this CCK satiety response, we administered the MC3/MC4R antagonist, SHU9119, into the PVN of rats before ip CCK-8 administration. PVN administration of SHU9119 attenuated the ability of CCK-8 to reduce 30-min food intake by 20%. To determine whether MC4R are expressed by PVN neurons that project directly to hindbrain nuclei involved in the satiety response to ip CCK-8, the retrograde tracer fluorescent cholera toxin subunit B was injected into the nucleus tractus solitarius (NTS) of the hindbrain. After 4 days, labeled PVN neurons were collected by laser capture microdissection and found to express MC4R mRNA by quantitative RT-PCR analysis. These data provide evidence for a neuroanatomical link between hypothalamic melanocortin signaling in the PVN and NTS neurons that regulate food intake. These findings highlight the contribution of melanocortin signaling in the PVN toward regulating the satiety effects of CCK-8 while acknowledging that melanocortin-dependent pathways in other brain regions and/or melanocortin-independent mechanisms are also important in this mechanism.

Fig. 1.

Effect of intraperitoneal (ip) administration of CCK-8 (0, 0.75, 2.5, 7.5 nmol/kg lean body mass) on 30-min food intake in age- and sex-matched MC4R^−/− and wild-type mice. Data represent means ± SE. Age- and sex-matched mice deprived of food for 18 h received an injection of CCK-8 or vehicle (0.1% BSA) in randomized order immediately before the start of the dark cycle. Food intake was measured 30 min after wild-type (n = 6–8) and MC4R^−/− mice (n = 12–13) received injections of CCK-8 or vehicle and access to food at the onset of the dark cycle. A: response to CCK-8 in wild-type mice. B: response to CCK-8 in MC4R^−/− mice. *P < 0.05 vs. vehicle (VEH). †P < 0.01 vs. VEH.

Fig. 2.

Effect of paraventricular nucleus (PVN) administration of SHU9119 on ip CCK-induced inhibition of food intake. Data represent means ± SE. SHU9119 or SHU9119 vehicle (saline) was administered 1 h before a dose of CCK-8 or CCK-8 vehicle given ip (0.1% BSA, saline). Food intake was measured 30 min after animals (n = 17) received ip injections of CCK-8 or vehicle and access to food at the onset of the dark cycle. *P < 0.05 vs. CCK-8.

Fig. 3.

Representative photomicrograph taken of injection sites within the PVN. The arrow represents the ventral most portion of the injection sites on both sides of the PVN.

Fig. 4.

Histological analysis of injection sites within the PVN. Distribution of injection sites from animals that had guide cannulas directed at the PVN. Data from an animal were excluded if its injection site extended beyond the boundary of the PVN.

Fig. 5.

Representative image visualized using the Arcturus AutoPix Fluorescent LCM System of parvocellular PVN (pPVN) neurons labeled with Alexa 488-conjugated cholera toxin subunit B (CTB) that project to the nucleus of solitary tract (NTS). A: Alexa 488-conjugated CTB-labeled cells in pPVN neurons as visualized on the Arcturus AutoPix Fluorescent LCM System. B: Alexa 488-conjugated CTB-labeled cells targeted for collection (before collection). C: Alexa 488-conjugated CTB-labeled cells collected for analysis.

Fig. 6.

Relative mRNA levels of MC4R, oxytocin, and corticotropin-releasing hormone (CRH) on pPVN neurons that project to the NTS. Data represent means ± SE. A: relative MC4R mRNA levels following analysis of Alexa 488-conjugated CTB-labeled pPVN neurons by quantitative RT-PCR, with the data normalized to GAPDH mRNA levels (44). Relative MC4R mRNA levels in CTB-labeled pPVN neurons were compared with levels in suprachiasmatic nucleus (SCN) as negative control. B: relative oxytocin- and CRH-mRNA following analysis of Alexa 488-conjugated CTB-labeled pPVN neurons by quantitative RT-PCR, with the data normalized to GAPDH mRNA levels. Relative oxytocin- and CRH-mRNA levels were compared with levels in SCN as a negative control. †P < 0.01 vs. SCN.