Intrahypothalamic pituitary adenylate cyclase-activating polypeptide regulates energy balance via site-specific actions on feeding and metabolism

Abstract

Numerous studies have demonstrated that both the hypothalamic paraventricular nuclei (PVN) and ventromedial nuclei (VMN) regulate energy homeostasis through behavioral and metabolic mechanisms. Receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are abundantly expressed in these nuclei, suggesting PACAP may be critical for the regulation of feeding behavior and body weight. To characterize the unique behavioral and physiological responses attributed to select hypothalamic cell groups, PACAP was site-specifically injected into the PVN or VMN. Overall food intake was significantly reduced by PACAP at both sites; however, meal pattern analysis revealed that only injections into the PVN produced significant reductions in meal size, duration, and total time spent eating. PACAP-mediated hypophagia in both the PVN and VMN was abolished by PAC1R antagonism, whereas pretreatment with a VPACR antagonist had no effect. PACAP injections into the VMN produced unique changes in metabolic parameters, including significant increases in core body temperature and spontaneous locomotor activity that was PAC1R dependent whereas, PVN injections of PACAP had no effect. Finally, PACAP-containing afferents were identified using the neuronal tracer cholera toxin subunit B (CTB) injected unilaterally into the PVN or VMN. CTB signal from PVN injections was colocalized with PACAP mRNA in the medial anterior bed nucleus of the stria terminalis, VMN, and lateral parabrachial nucleus (LPB), whereas CTB signal from VMN injections was highly colocalized with PACAP mRNA in the medial amygdala and LPB. These brain regions are known to influence energy homeostasis perhaps, in part, through PACAP projections to the PVN and VMN.

Keywords: activity; feeding; hypothalamus; rat; temperature.

Fig. 1.

Injection spread of biotin-labeled pituitary adenylate cyclase-activating polypeptide (PACAP). Staining for biotin-labeled PACAP (far right) accompanied by adjacent cresyl violet staining (far left) show that spread of injectate is contained within the boundaries of the PVN (A; Bregma −1.8 mm) or VMN (B; Bregma −3.0 mm). Schematic image corresponding to the level of injection site is shown in the middle, adapted from The Rat Brain in Stereotaxic Coordinates 6th Edition (48). PVN, paraventricuar nuclei; AHA, anterior hypothalamic area; VMN, ventromedial nuclei.

Fig. 2.

PACAP injections into the PVN or VMN both decrease feeding behavior and body weight. Bilateral 50 pmol PACAP injections (0.25 μl/side) into the PVN (A) or VMN (B) result in significantly reduced cumulative food intake 3 and 5 h postinjection (left), accompanied by reduction in body weight 24 h later (right). Data are expressed as means ± SE. *P < 0.05 vs. saline.

Fig. 3.

In situ hybridization of hypothalamic PACAP receptor mRNA. mRNA expression of the VPAC2R (middle) and PAC1R (far right) in the hypothalamic PVN (A; Bregma −1.8 mm), and VMN (B; Bregma −3.0 mm) accompanied by adjacent sections stained with cresyl violet depicting the corresponding hypothalamic anatomy in far left.

Fig. 4.

PACAP effects on feeding are mediated by PAC1R. In both PVN (A) and VMN (B), PACAP-induced hypophagia (50 pmol/side) is prevented by PAC1R antagonist PACAP(6–38) (500 pmol/side; right), but not the VPACR antagonist vasoactive intestinal polypeptide (VIP)(6–28) (500 pmol/side; left). Data are expressed as means ± SE. *P < 0.05 vs. saline.

Fig. 5.

VMN but not PVN injections of PACAP increase thermogenesis and activity. Time course of core body temperature (A) and 3 h temperature change (Δ; B) show a significant increase following PACAP injections (50 pmol) into the VMN; however, injections into the PVN do not affect temperature. Time course for cumulative activity (C) and area under the curve (AUC; D) analysis depict a similar significant increase only in animals receiving injections of PACAP into the VMN. Data are expressed as means ± SE. *P < 0.05 vs. saline.

Fig. 6.

PACAP-mediated increases in temperature by the VMN are PAC1R dependent. A and B: PAC1R antagonist PACAP(6–38) (500 pmol/side) attenuates the time course and 3-h temperature change (Δ) of PACAP-induced thermogenesis (50 pmol/side). C: interscapular brown adipose tissue (iBAT) triglyceride (TG) content was significantly decreased 3 h following injections of PACAP into the VMN. D: VMN injections of VPACR agonist VIP (50 pmol/side) did not alter the time course of core body temperature, whereas PAC1R-specific agonist maxadilan (50 pmol/side) caused significant increases in the time course (E) and 3 h temperature change (Δ; F) in core body temperature similar to PACAP injections. Data are expressed as means ± SE. *P < 0.05 vs. saline group; #P < 0.05 vs. PACAP group.

Fig. 7.

PACAP-mediated increases in activity by the VMN are PAC1R dependent. A: time course of PACAP-induced spontaneous locomotor activity (50 pmol/side) is blocked by PAC1R antagonist PACAP(6–38) (500 pmol/side), shown by area AUC analysis (B). C: VMN injections of VPACR agonist VIP (50 pmol/side) did not alter the time course of activity, whereas PAC1R-specific agonist maxadilan (50 pmol/side) caused significant increases in the time course of locomotor activity shown by AUC analysis similar to PACAP injections (D and E). Data are expressed as means ± SE. *P < 0.05 vs. saline group.

Fig. 8.

PACAP-containing afferents of the PVN include the anterior medial bed nucleus of the stria terminalis (BNST), VMN, and lateral parabrachial nucleus (LPB). Left: fluorescent in situ hybridization for VGLUT2 or PACAP identifying anatomic borders of nuclei and cell bodies. Middle: immunofluorescence for cholera toxin subunit B (CTB). Right: merged images. The majority of cells (∼90%) in the VMN and LPB projecting to the PVN were positive for PACAP, whereas ∼50% of cells projecting from the BNST were positive for PACAP. PVN injection site at ×10 (A–C); anterior medial BNST at ×10 (D–F) and ×40 (G–I); VMN at ×10 (J–L) and ×40 (M–O); LPB at ×10 (P–R) and ×40 (S–U). Boxes at left: area of subsequent high-magnification pictures; arrows, representative dual-labeled cells. 3V, 3rd ventricle; ac, anterior commissure.

Fig. 9.

PACAP-containing afferents of the VMN include the medial amygdala (MeA), and LPB. Left: fluorescent in situ hybridization for VGLUT2 or PACAP identifying anatomic borders of nuclei and cell bodies. Middle: immunofluorescence for CTB. Right: merged images. The majority of cells projecting to the VMN from the MeA and LPB were positive for PACAP (∼85–90%). VMN injection site at ×5 (A–C); MeA at ×10 (D–F) and ×40 (G–I); LPB at ×10 (J–L) and ×40 (M–O). Boxes at left: area of subsequent high-magnification pictures; arrows, representative dual-labeled cells. opt, optic tract.