PACAP intraperitoneal treatment suppresses appetite and food intake via PAC1 receptor in mice by inhibiting ghrelin and increasing GLP-1 and leptin

Abstract

Pituitary adenylate cyclase-activating peptide (PACAP) is expressed within the gastroenteric system, where it has profound physiological effects. PACAP was shown to regulate food intake and thermogenesis centrally; however, PACAP peripheral regulation of appetite and feeding behavior is unknown. Therefore, we studied PACAP's effect on appetite and food intake control by analyzing feeding behavior and metabolic hormones in PAC1-deficient (PAC1-/-) and age-matched wild-type (WT) mice intraperitoneally injected with PACAP1-38 or PACAP1-27 before the dark phase of feeding. Food intake and feeding behavior were analyzed using the BioDAQ system. Active ghrelin, glucagon-like peptide-1 (GLP-1), leptin, peptide YY, pancreatic polypeptide, and insulin were measured following PACAP1-38 administration in fasted WT mice. PACAP1-38/PACAP1-27 injected into WT mice significantly decreased in a dose-dependent manner cumulative food intake and reduced bout and meal feeding parameters. Conversely, PACAP1-38 injected into PAC1-/- mice failed to significantly change food intake. Importantly, PACAP1-38 reduced plasma levels of active ghrelin compared with vehicle in WT mice. In PAC1-/- mice, fasting levels of active ghrelin, GLP-1, insulin, and leptin and postprandial levels of active ghrelin and insulin were significantly altered compared with levels in WT mice. Therefore, PAC1 is a novel regulator of appetite/satiety. PACAP1-38/PACAP1-27 significantly reduced appetite and food intake through PAC1. In PAC1-/- mice, the regulation of anorexigenic/orexigenic hormones was abolished, whereas active ghrelin remained elevated even postprandially. PACAP significantly reduced active ghrelin in fasting conditions. These results establish a role for PACAP via PAC1 in the peripheral regulation of appetite/satiety and suggest future studies to explore a therapeutic use of PACAP or PAC1 agonists for obesity treatment.

Keywords: GLP-1; PAC1 receptor; appetite; ghrelin; leptin; pituitary adenylate cyclase-activating peptide.

Copyright © 2015 the American Physiological Society.

Figures

Fig. 1.

Pituitary adenylate cyclase-activating peptide (PACAP) variants PACAP1–38 and PACAP1–27 intraperitoneally (IP) injected at a dose of 100 nM, 1 μM, or 10 μM in 200 μl of saline induced a decrease in cumulative food intake in a dose-dependent manner during the dark phase of feeding in wild-type (WT) but not PAC1-deficient (PAC1−/−) mice. A: PACAP1–38 injection in WT mice significantly reduced cumulative food intake in a dose-dependent manner, beginning at 3 h with a 10 μM dose, at 4 h with a 1 μM dose, and at 8 h with a 100 nM dose. B: PACAP1–27 injection in WT mice significantly reduced manner cumulative food intake in a dose-dependent, beginning at 3 h with a 10 μM dose, at 4 h with a 1 μM dose, and at 8 h with a 100 nM dose. C: no significant reduction in food intake was observed following PACAP1–38 injection at 100 nM, 1 μM, or 10 μM dose in PAC1−/− mice. Data are means ± SE of 16 mice/group. **P < 0.01; ****P < 0.0001, 10 μM vs. vehicle. #P < 0.05; ##P < 0.01; ###P < 0.001; ####P < 0.0001, 1 μM vs. vehicle. ×P < 0.05; ××P < 0.01, 100 nM vs. vehicle.

Fig. 2.

Twenty-four-hour total food consumption was reduced in a dose-dependent manner following IP injection of either PACAP1–38 or PACAP1–27 before the dark phase in WT but not in PAC1−/− mice. A: PACAP1–38 injected in WT mice compared with vehicle. B: PACAP1–27 injected in WT mice compared with vehicle. C: PACAP1–38 injected in PAC1−/− mice compared with vehicle. Data are means ± SE of 16 mice/group. ****P < 0.0001, 10 μM vs. vehicle. ###P < 0.001; ####P < 0.0001, 1 μM vs. vehicle. ×P < 0.05; ××P < 0.01, 100 nM vs. vehicle.

Fig. 3.

PACAP1–38 and PACAP1–27 reduced food intake in a dose-dependent manner following IP injection before the dark phase of feeding in WT but not in PAC1−/− mice. A: PACAP1–38 injection in WT mice at 100 nM, 1 μM, and 10 μM reduced food intake in a dose-dependent manner 0–4 h postinjection compared with vehicle, and PACAP1–38 injected at the higher dose of 10 μM continued to show a reduced food intake for an additional 4–8 h postinjection. B: PACAP1–27 injection in WT mice at 1 and 10 μM reduced food intake in a dose-dependent manner 0–4 h and 4–8 h postinjection compared with vehicle. C: in PAC1−/− mice, IP injections of PACAP1–38 failed to cause significant alteration of food intake. Data are means ± SE of 16 mice/group. ****P < 0.0001, 10 μM vs. vehicle. #P < 0.05; ####P < 0.0001, 1 μM vs. vehicle. ××P < 0.01, 100 nM vs. vehicle.

Fig. 4.

Analysis of the feeding microstructure in the studied mice, using an automated episodic feeding monitoring system, showed that PACAP1–38 injection significantly altered all the examined feeding behavior parameters compared with vehicle. A: bout duration was reduced during the first 0–4 h and 4–8 h post PACAP1–38 injection. B: bout frequency was reduced during the first 0–4 h and 4–8 h post PACAP1–38 injection. C: time spent in feeding was also reduced during the first 0–4 h and 4–8 h post PACAP1–38 injection. D: meal size was decreased in the first0-4 h and 4–8 h post PACAP1–38 injection. E: time spent in meals was reduced 0–4 h post PACAP1–38 injection. F: total meal time was reduced 0–4 h post PACAP1–38 injection. G: meal duration was also reduced 0–4 h post PACAP1–38 injection. H: eating rate was reduced 0–4 h and 4–8 h post PACAP1–38 injection. Data are means ± SE of 16 mice/group. **P < 0.01; ***P < 0.001; ****P < 0.0001, 10 μM vs. vehicle. #P < 0.05; ###P < 0.001; ####P < 0.0001, 1 μM vs. vehicle. ××××P < 0.0001, 100 nM vs. vehicle.

Fig. 5.

PACAP1–38 reduced plasma active ghrelin levels in fasted WT mice. Mice were fasted overnight before 1 μM of PACAP1–38 in 200 μl of saline was injected. Blood plasma was withdrawn 30 min postinjection. A: PACAP1–38 injected into fasted WT mice significantly reduced plasma active ghrelin compared with vehicle. B: percentage of ratio of plasma active ghrelin to total ghrelin. C–F: plasma levels of glucagon-like peptide-1 (GLP-1; C), insulin (D), glucagon (E), and peptide YY (PYY; F) were not significantly altered compared with vehicle. Data are means ± SE of 8 mice/group. *P < 0.05.

Fig. 6.

Differences in metabolic hormones in fasting and postprandial conditions between PAC1−/− and WT mice. In fasting conditions, mice were fasted overnight and plasma was withdrawn. In postprandial conditions, mice were fasted overnight and refed for 30 min before plasma was withdrawn. Plasma levels of active ghrelin (A), percentage of active ghrelin/total ghrelin (B), GLP-1 (C), insulin (D), leptin (E), glucagon (F), and PYY (G) were assessed in PAC1−/− compared with WT mice. Data are means ± SE of 8 mice/group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.