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Selenoproteins are involved in the regulation of redox status, which affects several cellular processes, including cell survival and homeostasis. Considerable interest has arisen recently concerning the role of selenoproteins in the regulation of glucose metabolism. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein of the endoplasmic reticulum, is present at high levels in human and mouse pancreas as revealed by immunofluorescence and quantitative PCR. Confocal immunohistochemistry studies revealed that SelT is mostly confined to insulin- and somatostatin-producing cells in mouse and human islets. To elucidate the role of SelT in β-cells, we generated, using a Cre-Lox strategy, a conditional pancreatic β-cell SelT-knockout C57BL/6J mice (SelT-insKO) in which SelT gene disruption is under the control of the rat insulin promoter Cre gene. Glucose administration revealed that male SelT-insKO mice display impaired glucose tolerance. Although insulin sensitivity was not modified in the mutant mice, the ratio of glucose to insulin was significantly higher in the SelT-insKO mice compared with wild-type littermates, pointing to a deficit in insulin production/secretion in mutant mice. In addition, morphometric analysis showed that islets from SelT-insKO mice were smaller and that their number was significantly increased compared with islets from their wild-type littermates. Finally, we found that SelT is up-regulated by pituitary adenylate cyclase-activating polypeptide (PACAP) in β-pancreatic cells and that SelT could act by facilitating a feed-forward mechanism to potentiate insulin secretion induced by the neuropeptide. Our findings are the first to show that the PACAP-regulated SelT is localized in pancreatic β- and δ-cells and is involved in the control of glucose homeostasis.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid C-terminally alpha-amidated peptide that was first isolated 20 years ago from an ovine hypothalamic extract on the basis of its ability to stimulate cAMP formation in anterior pituitary cells (Miyata et al., 1989. PACAP belongs to the vasoactive intestinal polypeptide (VIP)-secretin-growth hormone-releasing hormone-glucagon superfamily. The sequence of PACAP has been remarkably well conserved during evolution from protochordates to mammals, suggesting that PACAP is involved in the regulation of important biological functions. PACAP is widely distributed in the brain and peripheral organs, notably in the endocrine pancreas, gonads, respiratory and urogenital tracts. Characterization of the PACAP precursor has revealed the existence of a PACAP-related peptide, the activity of which remains unknown. Two types of PACAP binding sites have been characterized: type I binding sites exhibit a high affinity for PACAP and a much lower affinity for VIP, whereas type II binding sites have similar affinity for PACAP and VIP. Molecular cloning of PACAP receptors has shown the existence of three distinct receptor subtypes: the PACAP-specific PAC1-R, which is coupled to several transduction systems, and the PACAP/VIP-indifferent VPAC1-R and VPAC2-R, which are primarily coupled to adenylyl cyclase. PAC1-Rs are particularly abundant in the brain, the pituitary and the adrenal gland, whereas VPAC receptors are expressed mainly in lung, liver, and testis. The development of transgenic animal models and specific PACAP receptor ligands has strongly contributed to deciphering the various actions of PACAP. Consistent with the wide distribution of PACAP and its receptors, the peptide has now been shown to exert a large array of pharmacological effects and biological functions. The present report reviews the current knowledge concerning the pleiotropic actions of PACAP and discusses its possible use for future therapeutic applications.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is known to counteract in vitro the deleterious effects of toxic agents on cerebellar granule cell survival and differentiation. The potent antiapoptotic action of PACAP is mediated through inhibition of caspase-3 activity; however, additional proteins are likely involved and remain to be identified. Two-dimensional gel electrophoresis analysis coupled with mass spectrometry characterization led to the identification of a protein, peroxiredoxin 2, which was induced after a 6-h treatment with PACAP. Western blot analysis confirmed the regulation of peroxiredoxin 2 by PACAP and revealed that this protein is induced by both cyclic AMP and protein kinase C stimulators. Inhibition of peroxiredoxin 2 expression, using two distinct small-interfering RNAs (siRNAs), reduced the effect of PACAP on caspase-3 activity and cerebellar granule cell survival. Peroxiredoxin 2 expression was also induced in vivo and in vitro by ethanol. Although ethanol and PACAP exert opposite effects on caspase-3 activity, inhibition of peroxiredoxin 2 expression, using siRNAs, only reduced the ability of PACAP to prevent ethanol-induced caspase-3 activity. Taken together, these data indicate that peroxiredoxin 2 is probably involved in the neurotrophic effect of PACAP and suggest that this protein may have a therapeutic potential for the treatment of some neurodegenerative diseases.

Cisplatin is a chemotherapeutic agent whose use is limited by side effects including neuropathies. In proliferating cells, toxic action of cisplatin is based on DNA interactions, while, in quiescent cells, it can induce apoptosis by interacting with proteins. In the present study, we compared cytotoxic mechanisms activated by cisplatin in primate and rodent neurons and in ovary cells in order to determine whether the anti-apoptotic peptide PACAP could selectively reduce neurotoxicity. In quiescent neurons, JNK and sphingomyelinase inhibitors blocked cisplatin-induced cell death. Toxicity was associated with DNA laddering, caspase-3 and -9 activations and Bax induction. These effects were prevented by PACAP. In proliferating cells, cisplatin activated caspase-8 but had no effect on caspase-9. PACAP exerted no protective effect. These data indicate that cisplatin activates distinct apoptotic pathways in quiescent neurons and proliferating cells and that PACAP may reduce neurotoxicity of cisplatin without affecting its chemotherapeutic efficacy.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic activities during cerebellar development, and a neuroprotective effect of PACAP has been demonstrated in pathological conditions such as stroke. However, all these data have been obtained in rodents, and neuroprotective effects of PACAP in primates remain unknown. Because of their evolutionary relationships with humans, monkeys represent powerful models for validating the therapeutic interest in PACAP. The objective of the present study was to characterize PACAP and its receptors in the cerebellum of two nonhuman primates. RT-PCR and in situ hybridization experiments revealed that PACAP is expressed in the cerebellum by Purkinje cells. Via immunohistochemistry, PACAP was detected in Purkinje cells and radial glial fibers. With regard to PACAP receptors, PAC1-R and VPAC1-R were detected by RT-PCR. In situ hybridization revealed a strong expression of PAC1-R and VPAC1-R in the granule cell layer (GCL), and VPAC1-R was also expressed in the Purkinje cell layer. A high density of PACAP binding sites was visualized in the GCL and the Purkinje cell layer. Competition studies indicated that, in the GCL, PACAP induced complete displacement of [(125)I]PACAP27 binding, whereas vasoactive intestinal polypeptide (VIP) was a weak competitor. In contrast, in the Purkinje cell layer, both PACAP and VIP displaced [(125)I]PACAP27 binding. Measurement of cAMP levels showed that PACAP is a powerful activator of adenylyl cyclase, whereas VIP is about 100-fold less potent. Altogether, these observations constitute the first demonstration of a functional PACAPergic system in monkey cerebellum. They strongly suggest that neuroprotective effects of PACAP can be transposed to primates, including human., ((c) 2007 Wiley-Liss, Inc.)

In the rodent cerebellum, PACAP is expressed by Purkinje neurons and PAC1 receptors are present on granule cells during both the development period and in adulthood. Treatment of granule neurons with PACAP inhibits proliferation, slows migration, promotes survival and induces differentiation. PACAP also protects cerebellar granule cells against the deleterious effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated through the cAMP/PKA signaling pathway and often involve the ERK MAPkinase. Caspase-3 is one of the key enzymes implicated in the neuroprotective action of PACAP but PACAP also inhibits caspase-9 activity and increases Bcl-2 expression. PACAP and functional PAC1 receptors are expressed in the monkey and human cerebellar cortex with a pattern of expression very similar to that described in rodents, suggesting that PACAP could also exert neurodevelopmental and neuroprotective functions in the cerebellum of primates including human.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits C2-ceramide-induced cell death through blockade of the mitochondrial apoptotic pathway in rat cerebellar granule neurones. However, the gene induction processes and transcription factors involved in the anti-apoptotic effect of PACAP remain unknown. Here, we show that PACAP and C2-ceramide activate activator protein-1 (AP-1) DNA binding in a dose- and time-dependent manner, but generate different AP-1 dimers. Thus, PACAP increased the proportion of c-Fos and Jun D while C2-ceramide increased c-Jun and reduced c-Fos in AP-1 complexes. In addition, PACAP strongly activated c-Fos gene expression while C2-ceramide markedly increased c-Jun phosphorylation. The effect of PACAP on c-Fos expression was blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor, U0126, while phosphorylation of c-Jun induced by C2-ceramide was abrogated by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid. Transfection of immature granule cells with c-Fos siRNA, which strongly reduced basal and PACAP-stimulated levels of the protein, totally prevented the stimulatory effect of PACAP on Bcl-2 expression. The present study demonstrates that AP-1 complexes containing c-Fos mediate the effect of PACAP on Bcl-2 gene expression in cerebellar granule neurones. Our data also indicate that different AP-1 dimers are associated with the pro-apoptotic effect of C2-ceramide and the anti-apoptotic effect of PACAP.

During brain development, cells that fail to reach their final destination or to establish proper connections are eliminated. It has been shown that the proinflammatory cytokine second messenger ceramides and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) play pivotal roles in the histogenesis of the cerebellum. However, little is known regarding the effects of these two factors on cerebellar granule cell migration. We have found that PACAP prevents the effects of C2-ceramide on granule cell motility and neurite outgrowth. These actions are attributable to opposite effects on actin distribution, tubulin polymerization, and Tau phosphorylation. These data suggest that PACAP and factors inducing ceramide production may control granule cell migration during cerebellar development.

PACAP exerts neuroprotective effects during development, especially in the cerebellum where PAC1 receptor and ligand are both expressed. However, while previous studies using PACAP injections in postnatal animals defined trophic effects of exogenous peptide, the role of endogenous PACAP remains unexplored. Here, we used PAC1−/− mice to investigate the role of PACAP receptor signaling in postnatal day 7 cerebellum. There was no difference in DNA synthesis in the cerebellar EGL of PAC1−/− compared to wild type animals, assessed using thymidine incorporation and BrdU immunohistochemistry. In contrast, we found that a significant proportion of newly generated neurons were eliminated before they successfully differentiated in the granule cell layer. In aggregate, these results suggest that endogenous PACAP plays an important role in cell survival during cerebellar development, through the activation of the PAC1 receptor. [ABSTRACT FROM AUTHOR]