A potential role for hypothalamomedullary POMC projections in leptin-induced suppression of food intake

Abstract

Melanocortin-3/4 receptor ligands administered to the caudal brain stem potently modulate food intake by changing meal size. The origin of the endogenous ligands is unclear, because the arcuate nucleus of the hypothalamus and the nucleus of the solitary tract (NTS) harbor populations of proopiomelanocortin (POMC)-expressing neurons. Here we demonstrate that activation of hypothalamic POMC neurons leads to suppression of food intake and that this suppression is prevented by administration of a melanocortin-3/4 receptor antagonist to the NTS and its vicinity. Bilateral leptin injections into the rat arcuate nucleus produced long-lasting suppression of meal size and total chow intake. These effects were significantly blunted by injection of SHU-9119 into the fourth ventricle, although SHU-9119 increased meal size and food intake during the first, but not the second, 14-h observation period. Leptin effects on meal size and food intake were abolished throughout the 40-h observation period by injection of SHU-9119 into the NTS at a dose that by itself had no effect. Neuron-specific tracing from the arcuate nucleus with a Cre-inducible tract-tracing adenovirus in POMC-Cre mice showed the presence of labeled axons in the NTS. Furthermore, density of alpha-melanocyte-stimulating hormone-immunoreactive axon profiles throughout the NTS was decreased by approximately 70% after complete surgical transection of connections with the forebrain in the chronic decerebrate rat model. The results further support the existence of POMC projections from the hypothalamus to the NTS and suggest that these projections have a functional role in the control of food intake.

Fig. 1.

Suppression of food intake and meal size by leptin administration to the arcuate nucleus is attenuated by injection of the melanocortin 3/4 receptor (MC3R/MC4R) antagonist SHU-9119 (SHU) into the 4th ventricle (4V). Leptin (30 pmol/side) or saline (Sal) was injected bilaterally into the arcuate nucleus 1 h after administration of SHU-9119 (100 pmol) or saline into the 4th ventricle. A: cumulative food intake throughout the 40-h observation period. Horizontal gray bars indicate dark periods. B and C: food intake during the 2 dark periods (2–14 h and 26–38 h, including 2 h before dark onset). D and E: meal size and meal frequency during the 2 dark periods. *P < 0.05, based on multiple comparisons after ANOVA (A) or ANOVA with pairwise comparisons of differences between saline and leptin with and without SHU-9119 pretreatment (B–E). #P < 0.05, based on ANOVA with pairwise comparisons of differences between saline and SHU-9119 pretreatment with and without arcuate leptin injection.

Fig. 2.

Suppression of food intake and meal size by leptin administration to the arcuate nucleus is abolished by injection of the MC3R/MC4R antagonist SHU-9119 into the nucleus of the solitary tract (NTS). Leptin (30 pmol/side) or saline was injected bilaterally into the arcuate nucleus 1 h after administration of SHU-9119 (20 pmol) directly into the NTS. A: cumulative food intake throughout the 40-h observation period. Horizontal gray bars indicate dark periods. B and C: food intake during the 2 dark periods (0–14 h and 24–38 h, including 2 h before dark onset). D and E: meal size and meal frequency during the 2 dark periods. *P < 0.05, based on multiple comparisons after ANOVA (A) or pairwise comparisons of mean differences between saline and leptin with and without SHU-9119 pretreatment (B–E). #P < 0.05, based on ANOVA with pairwise comparisons of mean differences between saline and SHU-9119 pretreatment with and without arcuate leptin injection.

Fig. 3.

Leptin injection sites in the arcuate nucleus. A: histological verification of typical bilateral cannula tracks in the arcuate nucleus used to determine injector tip location. B: injection sites (●) for bilateral arcuate nucleus injections in rats with 4th ventricular administration of SHU-9119. C: injection sites for bilateral arcuate nucleus injections in rats with NTS administration of SHU-9119. Sites are superimposed on images from the stereotaxic atlas of Paxinos (26).

Fig. 4.

Neuron-specific tracing of arcuate proopiomelanocortin (POMC) neurons reveals direct projections to the NTS. Arcuate POMC neurons in POMC-Cre transgenic mice were selectively induced to express farnesylated green fluorescent protein (GFP) by 2 injections of 300 nl of local Cre-inducible adenovirus. Confocal image stacks (10 × 1.5 μm) show unilateral injection site in the arcuate nucleus with labeled neurons (A) and labeled axon profiles at different rostrocaudal levels of the NTS (B–D). ap, Area postrema; cc, central canal; dmnX, dorsal motor nucleus; ts, tractus solitarius; 3V, 3rd ventricle. Scale bar: 120 μm (A), 200 μm (B and D), and 150 μm (C).

Fig. 5.

Transection of all neural connections at midbrain level abolishes the majority of α-melanocyte-stimulating hormone (α-MSH) immunoreactive axon profiles in the NTS. α-MSH-immunoreactive fibers at 3 rostrocaudal levels of the NTS are shown in intact (A–C) and midcollicular decerebrate (D–F) rats 8 wk after decerebration. Large-caliber, beaded, varicose axon profiles in intact rats are completely absent in decerebrate rats. Only very fine, dustlike labeling was found in decerebrate rats. Scale bar: 150 μm for all images. See Fig. 4 legend for definition of abbreviations.

Fig. 6.

Quantitative assessment of relative contribution of forebrain and caudal brain stem to α-MSH innervation of the NTS. Densitometric analysis of α-MSH immunoreactivity at 3 rostrocaudal levels of the NTS of intact and decerebrate rats. α-MSH immunoreactivity is significantly reduced (*P < 0.01, based on ANOVA) by >70% at all 3 levels. AP, area postrema.