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. 2022 Mar 1;322(3):E199-E210.
doi: 10.1152/ajpendo.00320.2021. Epub 2022 Jan 10.

Pituitary adenylate cyclase-activating polypeptide receptor activation in the hypothalamus recruits unique signaling pathways involved in energy homeostasis

Brian Maunze  1 , Katherine Wood Bruckner  1 , Nikhil Nilesh Desai  1 , Christopher Chen  1 , Fanghong Chen  1 , David Baker  1 , SuJean Choi  1
Affiliations

Pituitary adenylate cyclase-activating polypeptide receptor activation in the hypothalamus recruits unique signaling pathways involved in energy homeostasis

Brian Maunze et al. Am J Physiol Endocrinol Metab. .

Abstract

Pituitary adenylate cyclase activating polypeptide (PACAP) exerts pleiotropic effects on ventromedial nuclei (VMN) of the hypothalamus and its control of feeding and energy expenditure through the type I PAC1 receptor (PAC1R). However, the endogenous role of PAC1Rs in the VMN and the downstream signaling responsible for PACAP's effects on energy balance are unknown. Numerous studies have revealed that PAC1Rs are coupled to both Gαs/adenylyl cyclase/protein kinase A (Gαs/AC/PKA) and Gαq/phospholipase C/protein kinase C (Gαq/PLC/PKC), while also undergoing trafficking following stimulation. To determine the endogenous role of PAC1Rs and downstream signaling that may explain PACAP's pleiotropic effects, we used RNA interference to knockdown VMN PAC1Rs and pharmacologically inhibited PKA, PKC, and PAC1R trafficking. Knocking down PAC1Rs increased meal sizes, reduced total number of meals, and induced body weight gain. Inhibition of either PKA or PKC alone in awake male Sprague-Dawley rats, attenuated PACAP's hypophagic and anorectic effects during the dark phase. However, PKA or PKC inhibition potentiated PACAP's thermogenic effects during the light phase. Analysis of locomotor activity revealed that PKA inhibition augmented PACAP's locomotor effects, whereas PKC inhibition had no effect. Finally, PACAP administration in the VMN induces surface PAC1R trafficking into the cytosol which was blocked by endocytosis inhibitors. Subsequently, inhibition of PAC1R trafficking into the cytosol attenuated PACAP-induced hypophagia. These results revealed that endogenous PAC1Rs uniquely engage PKA, PKC, and receptor trafficking to mediate PACAP's pleiotropic effects in VMN control of feeding and metabolism.NEW & NOTEWORTHY Endogenous PAC1 receptors, integral to VMN management of feeding behavior and body weight regulation, uniquely engage PKA, PKC, and receptor trafficking to mediate the hypothalamic ventromedial nuclei control of feeding and metabolism. PACAP appears to use different signaling mechanisms to regulate feeding behavior from its effects on metabolism.

Keywords: PAC1R; PACAP; energy expenditure; feeding; rat.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
AAV8-mediated transduction of VMN cells with PAC1R shRNA decreases PAC1R protein expression. A: ×10 stitched confocal photomicrograph of VMN cells 21 days after transduction with AAV8 containing control or PAC1R-shRNA (scale bar = 100 µm). Inset is a ×20 magnification of the injection area (scale bar = 50 µm). B: representative Western blots and quantification of PAC1R protein expression in VMN tissue from Sprague-Dawley rats injected with AAV8 nonsilencing control (NSc) or PAC1R shRNA. Data are expressed as means ± SE, *P < 0.05 compared with control (n = 8 rats/group). ARC = arcuate nucleus, 3V = third ventricle; PAC1R, pituitary adenylate cyclase receptor; VMN, ventromedial nuclei.
Figure 2.
Figure 2.
Knocking down PAC1Rs in the VMN increases daily food intake and cumulative body weight change. Knocking down VMN PAC1Rs increased daily food intake by day 12 (A) and increased body weight by day 18 (B). Inset in A shows a significant increase in dark phase food take but not light phase feeding averaged over the 21-day period. Knocking down VMN PAC1Rs is sufficient to block PACAP-induced decreases in food intake (C) and body weight (D). Data are expressed as means ± SE, *P < 0.05 compared with control (n = 8 rats/group). NSc, nonsilencing control; PAC1R, pituitary adenylate cyclase receptor; PACAP, pituitary adenylate cyclase-activating polypeptide; VMN, ventromedial nuclei.
Figure 3.
Figure 3.
Blocking PKA signaling with KT5750 blocks PACAP-dependent decreases in food intake and attenuates PACAP-induced decrease in body weight. A: blocking PKA signaling with KT5720 reverses PACAP-induced suppression of food intake from 5 h postinjection lasting up to 8 h. B: KT5720 attenuated PACAP-induced reduction in body weight 24 h following injection. KT5720 potentiates PACAP’s thermogenic (C) and locomotor effects (D). Data are expressed as means ± SE, *P < 0.05 (treatment vs. control), #P < 0.05 (PACAP vs. PACAP + KT5720); n = 6 rats/group. PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A.
Figure 4.
Figure 4.
Blocking PKC with GF109203X transiently reduces PACAP’s hypophagic and anorectic effects. A: blocking PKC with GF109203X attenuates PACAP-induced decrease in food intake for 3 h postinjection. B: GF109203X blocked PACAP-induced decrease in body weight 24 h following injection. GFX109203X potentiates PACAP-dependent increase in core body temperature (C) but not spontaneous locomotor activity (D). Data are expressed as means ± SE, *P < 0.05 (PACAP vs. control), #P < 0.05 (PACAP vs. PACAP + GF109203X); n = 6 rats/group. PACAP, pituitary adenylate cyclase-activating polypeptide; PKC, protein kinase A.
Figure 5.
Figure 5.
Inhibiting surface PAC1R trafficking attenuates PACAP-induced decrease in food intake and exaggerates PACAP-induced decrease in body weight. A and B: Western blot analysis showing PACAP promotes PAC1R trafficking from the membrane to the cytosol, which can be blocked by Dyngo-4a/Pitstop 2 and measured at 30 mins following PACAP injection. C: Dyngo-4a/Pitstop 2, dynamin and clathrin-dependent endocytosis inhibitors, briefly attenuate PACAP-induced suppression of food intake for 5 h postinjection. D: Dyngo-4a/Pitstop 2 does not prevent PACAP-induced reduction in body weight at 24 h. Data are expressed as means ± SE, *P < 0.05 (PACAP vs. control), #P < 0.05 (PACAP vs. PACAP+Dyngo-4a/Pitstop 2); n = 8 rats/group. PACAP, pituitary adenylate cyclase-activating polypeptide; PAC1R, pituitary adenylate cyclase receptor.
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