Pituitary adenylate cyclase-activating polypeptide (PACAP) has been conserved remarkably during evolution and is widely expressed in the mammalian brain. In Drosophila, mutation of the PACAP homologue results in behavioral defects, including impaired olfaction-associated learning and changes in ethanol sensitivity. Here, we report the generation of mice lacking the PACAP gene (PACAP ؊/؊ ). PACAP ؊/؊ mice were born in the expected Mendelian ratios but had a high early-mortality rate. The surviving adult PACAP ؊/؊ mice displayed remarkable behavioral changes; they exhibited hyperactive and explosive jumping behaviors in an open field, increased exploratory behavior, and less anxiety in the elevated plus maze, emergence, and novel-object tests. Analysis of PACAP ؊/؊ mice brains revealed that the serotonin metabolite 5-hydroxyindoleacetic acid was slightly decreased in the cortex and striatum compared with wild-type mice. The present study provides evidence that PACAP plays a previously uncharacterized role in the regulation of psychomotor behaviors. P ituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide (VIP)͞ secretin͞glucagon family of peptides and exists in two amidated forms, PACAP38 and PACAP27, that share an identical 27-aa N terminus and are alternatively processed from a 176-aa precursor called preproPACAP (1, 2). The primary structure of PACAP38 has been conserved significantly during evolution from protochordates to mammals, suggesting that the peptide exerts important activities throughout the vertebrate phylum (1, 2). In Drosophila, recent molecular cloning and transgenic rescue experiments in the memory-mutant amnesiac, which has behavioral defects that include impaired olfaction-associated learning and changes in ethanol sensitivity, demonstrated that the amnesiac gene encodes a neuropeptide homologous to vertebrate PACAP (3, 4). In addition, mammalian PACAP activated both the cAMP and Ras͞Raf signal-transduction pathways in Drosophila neurons, suggesting a neuromodulatory role of amnesiac (Drosophila PACAP) in specific neuronal populations (5). In mammals, PACAP occurs in neuronal elements, where it acts as a pleiotropic neuropeptide via three heptahelical G protein-linked receptors-one PACAP-specific (PAC 1 ) receptor and two receptors that it shares with VIP (VPAC 1 and VPAC 2 ). PACAP stimulates several different signaling cascades in neurons, leading to the activation of adenylate cyclase, phospholipase C, and mitogen-activated protein kinase and the mobilization of calcium (1, 2, 6). Histochemical studies have shown that PACAP immunoreactivity is observed in several brain regions, including the dopamine (DA) and serotonin (5-HT) systems, with high concentrations found in the nucleus accumbens, hypothalamus, amygdala, substantia nigra, and dorsal raphe (7-9). PAC 1 receptor also is expressed throughout the target areas of both the mesocorticolimbic and nigrostriatal DA systems as well as 5-HT system (10). In addition, VPAC 1 and VPAC 2 recepto...

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the vasoactive intestinal peptide/secretin/glucagon family, stimulates insulin secretion from islets in a glucose-dependent manner at femtomolar concentrations. To assess PACAP's pancreatic function in vivo, we generated transgenic mice overexpressing PACAP in the pancreas under the control of human insulin promoter. Northern blot and immunohistochemical analyses showed that PACAP is overexpressed in pancreatic islets, specifically in transgenic mice. Plasma glucose and glucagon levels during a glucose tolerance test were not different between PACAP transgenic mice and nontransgenic littermates. However, plasma insulin levels in transgenic mice were higher after glucose loading. Also, increases of streptozotocin-induced plasma glucose were attenuated in transgenic compared with nontransgenic mice. Notably, an increase in 5-bromo-2-deoxyuridine-positive ␤-cells in the streptozotocin-treated transgenic mice was observed but without differences in the staining patterns by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Morphometric analysis revealed that total islet mass tends to increase in 12-month-old transgenic mice but showed no difference between 12-weekold transgenic and nontransgenic littermates. This is the first time that PACAP has been observed to play an important role in the proliferation of ␤-cells. Diabetes

Abstract:Pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression was analyzed in PC12 cells. PC12 cells transfected with a PACAP promoter-luciferase reporter construct were utilized to investigate the effects of PACAP, either alone or in combination with nerve growth factor (NGF), on PACAP transcriptional response. PACAP induced transcription from the PACAP promoter through PACAP type I receptor (PAC 1 receptor). PACAP gene transcription was also induced by NGF. Simultaneous treatment with PACAP and NGF resulted in a synergistic transcriptional response that was more than three times the predicted response, based on a simple additive effect of both agents. This synergism in transcriptional response paralleled the PACAP mRNA levels, as determined by RT-PCR and northern blotting. The level of PACAP mRNA peaked 3 h after stimulation and gradually returned to basal levels by 48 h. PC12 cells are known to express predominantly the hop isoform of the PAC 1 receptor, which positively couples to both adenylate cyclase and phospholipase C. To determine the role of the cyclic AMP and protein kinase C pathways in PACAP gene expression, the effects of forskolin and phorbol 12-myristate 13-acetate (PMA) were then examined. PMA did not alter PACAP mRNA levels but enhanced forskolin-induced PACAP mRNA expression. Down-regulation of protein kinase C blocked the ability of PACAP to stimulate PACAP mRNA expression. The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) inhibitor PD98059 also blocked the PACAP mRNA expression induced by either PACAP or NGF but not that induced by a combination of PACAP and NGF. These results suggest that PACAP stimulates the PACAP gene expression in PC12 cells at least in part through activation of adenylate cyclase and protein kinase C signaling pathways and that the ERK1/2 cascade is involved in PACAP and NGF-induced PACAP gene expression, although redundant signaling pathways may also be involved. The present finding showing that PACAP in combination with NGF causes a synergistic increase in PACAP gene expression in PC12 cells supports the idea that PACAP acts as an autocrine regulatory factor. Key Words: Pituitary adenylate cyclaseactivating polypeptide-Promoter activity-mRNANerve growth factor.

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Pituitary adenylate cyclase-activating polypeptide (PACAP) is an intraislet neuropeptide and shares insulinotropic and insulinsensitizing properties with glucagon-like peptide-1 (GLP-1); however, the pathophysiological significance of PACAP in diabetes remains largely unknown. To assess this, we crossed our recently developed transgenic mice overexpressing PACAP in pancreatic ␤-cells (Tg/ϩ), with lethal yellow agouti (KKA y ) mice (A y /ϩ), a genetic model for obesity-diabetes, and examined the metabolic and morphological phenotypes of F 1 animals. Tg/ϩ mice with the A y allele (Tg/ϩ:A y /ϩ) developed maturity-onset obesity and diabetes associated with hyperglycemia, hyperlipidemia, and hyperphagia, similar to those of A y /ϩ mice, but hyperinsulinemia was significantly ameliorated in Tg/ϩ:A y /ϩ mice. Although A y /ϩ mice exhibited a marked increase in islet mass resulting from hyperplasia and hypertrophy, this increase was significantly attenuated in Tg/ϩ:A y /ϩ mice. Size frequency distribution analysis revealed that the very large islets comprising one-fourth of islets of A y /ϩ mice were selectively reduced in Tg/ϩ:A y /ϩ mice. Because functional defects have been demonstrated in the large islets of obese animal models, together these findings suggest that PACAP regulates hyperinsulinemia and the abnormal increase in islet mass that occurs during the diabetic process.PACAP, which exists in two molecular forms, either with 27 (PACAP27) or 38 (PACAP38) amino acid residues, belongs to the vasoactive intestinal polypeptide/secretin/glucagon superfamily (Arimura, 1998;Vaudry et al., 2000). Some members of this group, such as the gastrointestinal hormones glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide, stimulate ␤-cell growth, differentiation, and cell survival, in addition to their well documented nutrient-stimulated secretion of insulin (so-called "incretin effect") (Kieffer and Habener, 1999;Pospisilik et al., 2003). GLP-1 and glucose-dependent insulinotropic polypeptide are secreted postprandially from the L-cells of the lower small intestine and from the K-cells of the upper small intestine, respectively, and released into the circulatory system. In comparison, PACAP is a neuropeptide in pancreatic islets, where it may act as a parasympathetic and sensory neurotransmitter, and stimulate secretion of insulin in a glucosedependent manner (Kieffer and Habener, 1999;Sherwood et al., 2000;Filipsson et al., 2001). In addition, PACAP has been shown to be expressed in islet ␤-cells (Yada et al., 1997;Portela-Gomes et al., 2003). PACAP stimulates insulin secretion from insulin-secreting ␤-cell lines (Klinteberg et al., 1996;Straub and Sharp, 1996), isolated pancreas (Yokota et al., 1993;Yada et al., 1994;Bertrand et al., 1996), and intact animals (Fridolf et al., 1992). There is recent evidence to suggest that PACAP exerts not only insulinotropic effects but also insulin-sensitizing properties like GLP-1. Yada et al. (2000) showed that intraperitoneal administration of PACAP...