Philippe Brabet

PAC1 receptor–deficient mice display impaired insulinotropic response to glucose and reduced glucose tolerance

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a ubiquitous neuropeptide of the vasoactive intestinal peptide (VIP) family that potentiates glucose-stimulated insulin secretion. Pancreatic beta cells express two PACAP receptor subtypes, a PACAP-preferring (PAC1) and a VIP-shared (VPAC2) receptor. We have applied a gene targeting approach to create a mouse lacking the PAC1 receptor (PAC1(-/-)). These mice were viable and normoglycemic, but exhibited a slight feeding hyperinsulinemia. In vitro, in the isolated perfused pancreas, the insulin secretory response to PACAP was reduced by 50% in PAC1(-/-) mice, whereas the response to VIP was unaffected. In vivo, the insulinotropic action of PACAP was also acutely reduced, and the peptide induced impairment of glucose tolerance after an intravenous glucose injection. This demonstrates that PAC1 receptor is involved in the insulinotropic action of the peptide. Moreover, PAC1(-/-) mice exhibited reduced glucose-stimulated insulin secretion in vitro and in vivo, showing that the PAC1 receptor is required to maintain normal insulin secretory responsiveness to glucose. The defective insulinotropic action of glucose was associated with marked glucose intolerance after both intravenous and gastric glucose administration. Thus, these results are consistent with a physiological role for the PAC1 receptor in glucose homeostasis, notably during food intake.

Dissociation between Light-Induced Phase Shift of the Circadian Rhythm and Clock Gene Expression in Mice Lacking the Pituitary Adenylate Cyclase Activating Polypeptide Type 1 Receptor

The circadian clock located in the suprachiasmatic nucleus (SCN) organizes autonomic and behavioral rhythms into a near 24 hr time that is adjusted daily to the solar cycle via a direct projection from the retina, the retinohypothalamic tract (RHT). This neuronal pathway costores the neurotransmitters PACAP and glutamate, which seem to be important for light-induced resetting of the clock. At the molecular level the clock genes mPer1 and mPer2 are believed to be target for the light signaling to the clock. In this study, we investigated the possible role of PACAP-type 1 receptor signaling in light-induced resetting of the behavioral rhythm and light-induced clock gene expression in the SCN. Light stimulation at early night resulted in larger phase delays in PACAP-type 1 receptor-deficient mice (PAC1−/−) compared with wild-type mice accompanied by a marked reduction in light-induced mPer1, mPer2, and c-fos gene expression. Light stimulation at late night induced mPer1 and c-fos gene expression in the SCN to the same levels in both wild type andPAC1−/− mice. However, in contrast to the phase advance seen in wild-type mice,PAC1−/− mice responded with phase delays after photic stimulation. These data indicate that PAC1 receptor signaling participates in the gating control of photic sensitivity of the clock and suggest thatmPer1, mPer2, and c-fosare of less importance for light-induced phase shifts at night.

Anti-inflammatory role in septic shock of pituitary adenylate cyclase-activating polypeptide receptor

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two mediators synthesized by immune cells, specially under inflammatory and antigen stimulation conditions. Reports have shown that neuropeptides attenuate the deleterious consequences of septic shock both by down-regulating the production of proinflammatory mediators and by stimulating the production of anti-inflammatory cytokines by activated macrophages. In this study, we used a knockout for the PACAP receptor (PAC1−/−) to demonstrate an important protective role for PAC1 receptor in endotoxic shock. Moreover, our results indicate that PAC1 receptor acts in vivo as an anti-inflammatory receptor, at least in part, by attenuating lipopolysaccharide (LPS)-induced production of proinflammatory IL-6, which appears to be the main cytokine regulating the expression of the majority of the acute phase protein genes, which are an important deleterious component of septic shock. Besides, our findings point to endogenously produced VIP and PACAP as participants of the natural anti-inflammatory machinery. Because VIP and PACAP are two attractive candidates for the development of therapies against acute and chronic inflammatory diseases, septic shock, and autoimmune diseases, this paper represents a contribution to the understanding of the mechanism of action of these anti-inflammatory agents.

Altered Social Behavior in Pituitary Adenylate Cyclase-Activating Polypeptide Type I Receptor-Deficient Mice

The olfactory bulb plays a critical role in odor discrimination and in processing olfactory cues controlling social behavior in mammals. Given that the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) is highly expressed in the olfactory bulb, we examined its role in regulating olfaction and social investigation. We found that olfactory detection of nonsocial stimuli was similar in PAC1-deficient mice and wild-type (WT) littermates. In contrast, PAC1-deficient mice displayed markedly abnormal social behaviors. PAC1-deficient mice exhibited a faster decrease in social investigation after repeated exposure to social cues or ovariectomized female urine compared with WT mice. Moreover, PAC1-deficient females exhibited delayed affiliative behavior when housed with novel males, and PAC1-deficient males displayed excessive sexual mounting toward both females and males as well as reduced aggression and increased licking and grooming toward intruder males. In aggregate, these results uncover PAC1 signaling as an important factor in the development and/or functioning of neural pathways associated with pheromone processing and the regulation of social interactions in mice. In turn, these studies raise the potential clinical relevance of PACAP signaling dysfunctions in neuropsychiatric disorders characterized by social reciprocity impairments such as autism spectrum disorders.

Mild deficits in mice lacking pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) performing on memory tasks

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory.

Mice lacking the PACAP type I receptor have impaired photic entrainment and negative masking

The retinohypothalamic tract (RHT) is a retinofugal neuronal pathway which, in mammals, mediates nonimage-forming vision to various areas in the brain involved in circadian timing, masking behavior, and regulation of the pupillary light reflex. The RHT costores the two neurotransmitters glutamate and pituitary adenylate cyclase activating peptide (PACAP), which in a rather complex interplay are mediators of photic adjustment of the circadian system. To further characterize the role of PACAP/PACAP receptor type 1 (PAC1) receptor signaling in light entrainment of the clock and in negative masking behavior, we extended previous studies in mice lacking the PAC1 receptor (PAC1 KO) by examining their phase response to single light pulses using Aschoff type II regime, their ability to entrain to non-24-h light-dark (LD) cycles and large phase shifts of the LD cycle (jet lag), as well as their negative masking response during different light intensities. A prominent finding in PAC1 KO mice was a significantly decreased phase delay of the endogenous rhythm at early night. In accordance, PAC1 KO mice had a reduced ability to entrain to T cycles longer than 26 h and needed more time to reentrain to large phase delays, which was prominent at low light intensities. The data obtained at late night indicated that PACAP/PAC1 receptor signaling is less important during the phase-advancing part of the phase-response curve. Finally, the PAC1 KO mice showed impaired negative masking behavior at low light intensities. Our findings substantiate a role for PACAP/PAC1 receptor signaling in nonimage-forming vision and indicate that the system is particularly important at lower light intensities.

Up-regulation of the PACAP type-1 receptor (PAC1) promoter by neurotrophins in rat PC12 cells and mouse cerebellar granule cells via the Ras/mitogen-activated protein kinase cascade.

The pituitary adenylate cyclase‐activating polypeptide type‐1 receptor (PAC1) has been involved in the survival and differentiation of neuroblasts during development. This study examined the effects of various neurotrophins on the activity of the mouse PAC1 promoter/luciferase reporter constructs in rat PC12 cells and in 8‐day‐old mouse cerebellar granule cells. In PC12 cells, both differentiating factors such as nerve growth factor (NGF) and mitogens such as epidermal growth factor (EGF) and insulin growth factor‐1 (IGF‐1) up‐regulated PAC1 promoter activity by 2–4‐fold in a concentration‐dependent manner. Although PACAP differentiated the PC12 cells, it had no effect on the PAC1 promoter and antagonized the stimulatory effect of NGF. In cerebellar granule cells, IGF‐1 and brain‐derived neurotrophic factor (BDNF) also stimulated the activity of the PAC1 promoter. NGF and IGF‐1 increased endogenous PAC1 mRNA levels, and the NGF‐induced up‐regulation is the result of an increase in transcription from PAC1 promoter instead of an increase in mRNA stability. The mitogen‐activated protein kinase (MAPK) kinase inhibitor, PD98059, prevented the transcriptional effects both in PC12 and cerebellar granule cells. Moreover, expression of dominant‐negative Ras protein in PC12 cells also prevented the NGF effect. Our results show that the PAC1 promoter can be up‐regulated by diverse neurotrophins via an MAPK‐dependent pathway and suggest a role for the Ras protein.

Characterization of intestinal receptors for VIP and PACAP in rat and in PAC1 receptor knockout mouse.

Abstract: The receptors for VIP and PACAP were characterized in vitro on rat ileal and colonic longitudinal smooth muscle with adherent myenteric ganglia. Colon strips from PAC1 receptor knockout and wildtype mice were also examined. VIP, PACAP‐38 and PACAP‐27 all caused concentration dependent relaxations. In rat ileum three different types of smooth muscle VIP/PACAP receptors were defined: (1) a PACAP‐27 preferring receptor coupled to apamin sensitive Ca2+‐dependent K+ channels, (2) a PAC1 receptor activated by both PACAP‐27 and PACAP‐38, and (3) a VIP specific receptor regulated by NPY. The receptors identified in rat colon were: (1) a PAC1 receptor localized on NO synthesizing neurones. Activation leads to increased NO production. (2) A smooth muscle PAC1 receptor. The responses elicited by both receptors were abolished by apamin. (3) A smooth muscle VIP specific receptor. PAC1 receptor knockout mice did not respond to PACAP‐27 or PACAP‐38, whereas VIP induced a relaxatory response indicating the presence of a VIP specific receptor. In wildtype mice all three peptides elicited relaxatory responses. Pharmacological characterization of intestinal VIP/PACAP receptors indicates the existence of receptors, such as a PACAP‐27 preferring receptor and a VIP specific receptor, distinct from those that have been cloned (VPAC1, VPAC2, and PAC1).

Induction of Type I PACAP Receptor Expression by the New Zinc Finger Protein Zac1 and p53

Abstract: We reported recently the cloning of the type I PACAP receptor by a functional expression cloning technique. Unexpectedly, we observed additional PACAP‐positive pools that turned out to encode the wild‐type form of the tumor suppressor gene p53 and the novel zinc finger protein Zac1, which regulates apoptosis and cell cycle arrest. 1 Both Zac1 and p53 caused, under transient or stably regulated expression, induction of the type I PACAP receptor by transcriptional mechanisms. Transactivation of the type I PACAP receptor gene by Zac1 and p53 points to a subtle balance between death promoting and protective mechanisms. The control of these processes is central to various physiological conditions ranging from development to senescence, whereas dysregulation may lead to overt pathological outcomes, notably cancer, immune deficiency syndromes, and neurodegenerative disorders.

Transcription of the mouse PAC1 receptor gene: cell-specific expression and regulation by Zac1

Regulations of the PACAP type 1 (PAC1) receptor expression have been described in the brain and the anterior pituitary. To understand the molecular mechanisms underlying mouse PAC1 gene regulation, we first mapped its transcription start sites (tss). PAC1 receptor RNA initiates from two major sites in embryos and adult tissues. Functional analysis revealed a basal promoter within the first 180 bp upstream of transcription start. Negative regulatory sequences upstream of this minimal promoter control the cell type-specific transcription of a luciferase reporter gene. Zac1, a zinc finger protein mainly expressed in the brain and the pituitary gland, binds to a GC-rich motif of the promoter regulatory elements. The Zac1 DNA binding site is required to positive and negative regulations of the promoter. Our findings provide bases for future studies on the regulatory elements controlling PAC1 gene transcription and demonstrate the PAC1 receptor promoter as a target of Zac1.