Critical role of arcuate Y4 receptors and the melanocortin system in pancreatic polypeptide-induced reduction in food intake in mice

Abstract

Background: Pancreatic polypeptide (PP) is a potent anti-obesity agent known to inhibit food intake in the absence of nausea, but the mechanism behind this process is unknown.

Methodology/principal findings: Here we demonstrate that in response to i.p. injection of PP in wild type but not in Y4 receptor knockout mice, immunostaining for the neuronal activation marker c-Fos is induced specifically in neurons of the nucleus tractus solitarius and the area postrema in the brainstem, notably in cells also showing immunostaining for tyrosine hydroxylase. Importantly, strong c-Fos activation is also detected in the arcuate nucleus of the hypothalamus (ARC), particularly in neurons that co-express alpha melanocyte stimulating hormone (alpha-MSH), the anorexigenic product of the proopiomelanocortin (POMC) gene. Interestingly, other hypothalamic regions such as the paraventricular nucleus, the ventromedial nucleus and the lateral hypothalamic area also show c-Fos induction after PP injection. In addition to c-Fos activation, PP injection up-regulates POMC mRNA expression in the ARC as detected by in situ hybridization. These effects are a direct consequence of local Y4 signaling, since hypothalamus-specific conditional Y4 receptor knockout abolishes PP-induced ARC c-Fos activation and blocks the PP-induced increase in POMC mRNA expression. Additionally, the hypophagic effect of i.p. PP seen in wild type mice is completely absent in melanocortin 4 receptor knockout mice.

Conclusions/significance: Taken together, these findings show that PP reduces food intake predominantly via stimulation of the anorexigenic alpha-MSH signaling pathway, and that this effect is mediated by direct action on local Y4 receptors within the ARC, highlighting a potential novel avenue for the treatment of obesity.

Figure 1. Pancreatic polypeptide (PP) injection induces a Y4 receptor-dependent increase in c-Fos immunoreactivity in the brainstem and hypothalamus.

(A,B and C) Photomicrograph of brains from wild type (WT) mice showing c-Fos immunoreactivity at 30 minutes after i.p. injection of saline. (D, E and F) Brains from wild type mice showing c-Fos immunoreactivity at 30 minutes after i.p. injection of PP. (G, H and I) Brains from Y4 receptor knockout mice (Y4 ^−/−) showing c-Fos immunoreactivity at 30 minutes after i.p. injection of PP. Scale bar for all panels  = 40 µm. NTS, nucleus tractus solitarus; AP, area postrema; ARC, arcuate nucleus of the hypothalamus; 3V, third cerebral ventricle.

Figure 2. Immunoreactivity for phosphorylated extracellular signal-regulated kinases 1 and 2 (P-Erk1/2) in the hypothalamus and co-expression of c-Fos and tyrosine hydroxylase (TH) immunoreactivity in the brainstem after PP injection.

(A) Brightfield micrograph showing P-Erk1/2 immunoreactivity in the arcuate nucleus of the hypothalamus (ARC) at 30 minutes after i.p. injection of PP in wild type mice. Scale bar = 200 µm. (B) Higher magnification of the boxed area from A. Scale bar  = 5 µm. (C, E) Fluorescence micrographs of the nucleus tractus solitarius (NTS) and area postrema (AP) respectively, 30 minutes after i.p. injection of PP. Scale bar  = 40 µm. (D, F) Higher magnifications of the NTS and AP, respectively, from the boxed areas in C and E. Scale bar  = 25 µm. White arrows indicate neurons positive for c-Fos immunoreactivity only (red fluorescence); white arrowheads indicate neurons positive for TH immunoreactivity only (green fluorescence); blue arrows indicate neurons double-labeled for c-Fos and TH. 4V, fourth cerebral ventricle; cc, central canal.

Figure 3. Pancreatic polypeptide (PP) injection induces c-Fos immunoreactivity in neurons positive for alpha melanocyte stimulating hormone (α-MSH) and glutamic acid decarboxylase 65 (GAD65) in the arcuate nucleus of the hypothalamus (ARC).

(A) c-Fos immunoreactivity, (B) α-MSH immunoreactivity, and (C) overlay of c-Fos and α-MSH immunoreactivity in neurons as indicated by white arrows at 30 minutes after i.p. injection of PP. Sale bar for A–C  = 25 µm. (D) Brightfield micrograph showing c-Fos and GAD65 immunoreactivity at 30 minutes after i.p. injection of PP. Scale bar  = 200 µm. (E) Higher magnification of the boxed area from D. Black arrows indicate neurons positive for c-Fos immunoreactivity only. These neurons are darkly stained. Black arrowheads indicate neurons positive for GAD65 immunoreactivity only. These neurons are lightly stained. Red arrows indicate neurons double-labeled for c-Fos and GAD65. The double staining on these neurons makes these neurons appear larger than the neurons positive only for c-Fos or CAG65 immunoreactvity. Scale bar  = 5 µm. 3V, third cerebral ventricle.

Figure 4. Effects of pancreatic polypeptide (PP) on the expression of proopiomelanocortin (POMC) and GAD65 mRNA in the arcuate nucleus of the hypothalamus (ARC).

Emulsion-dipped autoradiographs showing POMC mRNA at 30 minutes after i.p. injection of (A) saline or (C) PP, orGAD65 mRNA at 30 minutes after i.p. injection of (B) saline or (D) PP. Scale bar for all panels  = 25 µm. 3V, third cerebral ventricle.

Figure 5. Conditional deletion of Y4 receptors in the arcuate nucleus of the hypothalamus (ARC) alters pancreatic polypeptide (PP)-induced c-Fos immunoreactivity and POMC mRNA expression.

(A) Schematic representation of primer positions used for PCR verification of Y4 receptor gene knockout.The un-deleted gene is 3.4 kilobases, and after Cre-mediated Y4 receptor gene deletion a PCR product of 290 base pairs is produced. (B) Confirmation of Y4 receptor deletion (indicated by production of the 290 base pair PCR product) from DNA isolated from the hypothalamus (Hypo) but not from the cerebellum (Cer) of Y4 receptor conditional knockout (Y4F) mice injected with AAV-Cre recombinase into the ARC, or from mice not injected with AAV-Cre(Y4F Hypo). (C) Photomicrograph showing c-Fos immunoreactivity and (D+E) emulsion-dipped autoradiograph of POMC mRNA expression in the brain of a conditional Y4 receptor knockout mouse, 30 minutes after i.p. injection of PP. Mice received unilateral injection of (D) AAV-Cre or (E) AAV-empty 28 days prior to PP injection in order to induce local deletion of Y4 receptors by virally-delivered Cre-recombinase, the contra-lateral side (at left in C, D and E) was used as control. Scale bar  = 100 µm in C and 25 µm in D and E. 3V, third cerebral ventricle.

Figure 6. PP reduces food intake via a melanocortin 4 receptor-dependent pathway.

Twenty four (24) hour food intake (g/day) in wild type (WT) and melanocortin 4 receptor knockout (MC4R ^−/−) mice after i.p. injection of increasing doses of PP or saline vehicle. Data are means ± SEM of 6–8 mice per group. * P<0.05 versus vehicle-injected wild type mice or the comparison shown by horizontal bars.