We have previously demonstrated that ghrelin stimulates the cellular proliferation of cultured adult rat hippocampal neural stem cells (NSCs). However, little is known about the molecular mechanisms by which ghrelin regulates cell cycle progression. The purpose of this study was to investigate the potential effects of ghrelin on cell cycle regulatory molecules in cultured hippocampal NSCs. Ghrelin treatment increased proliferation assessed by CCK-8 proliferation assay. The expression levels of proliferating cell nuclear antigen and cell division control 2, well-known cell-proliferating markers, were also increased by ghrelin. Fluorescence-activated cell sorting analysis revealed that ghrelin promoted progression of cell cycle from G₀/G₁ to S phase, whereas this progression was attenuated by the pretreatment with specific inhibitors of MEK/extracellular signal-regulated kinase 1/2, phosphoinositide 3-kinase/Akt, mammalian target of rapamycin, and janus kinase 2/signal transducer and activator of transcription 3. Ghrelin-induced proliferative effect was associated with increased expression of E2F1 transcription factor in the nucleus, as determined by Western blotting and immunofluorescence. We also found that ghrelin caused an increase in protein levels of positive regulators of cell cycle, such as cyclin A and cyclin-dependent kinase (CDK) 2. Moreover, p27^KIP1 and p57^KIP2 protein levels were reduced when cell were exposed to ghrelin, suggesting downregulation of CDK inhibitors may contribute to proliferative effect of ghrelin. Our data suggest that ghrelin targets both cell cycle positive and negative regulators to stimulate proliferation of cultured hippocampal NSCs.

Exendin-4 is a naturally occurring more potent and stable analog of glucagon-like peptide-1 (GLP-1) that selectively binds at the GLP-1 receptor. It has been recently demonstrated that GLP-1 receptor stimulation preserves dopaminergic neurons in cellular and rodent models of Parkinson's disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic neurotoxicity in rodents; previous studies suggest that activated microglia actively participate in the pathogenesis of PD neurodegeneration. However, the role of microglia in the neuroprotective properties of exendin-4 is still unknown. Here, we show that, in the mouse MPTP PD model, systemic administration of exendin-4 significantly attenuates the loss of substantia nigra pars compacta (SNpc) neurons and the striatal dopaminergic fibers. Exendin-4 prevents MPTP-induced microglial activation in the SNpc and striatum, and the expression of matrix metalloproteinase-3. In addition, exendin-4 also suppressed MPTP-induced expression of pro-inflammatory molecules and tumor necrosis factor α and interleukin-1β. Our data indicate that exendin-4 may act as a survival factor for dopaminergic neurons by functioning as a microglia-deactivating factor and suggest that exendin-4 may be a valuable therapeutic agent for neurodegenerative diseases such as PD.

Only acylated ghrelin (AG) binds GH secretagog receptor 1a (GHS-R1a) and has central endocrine activities. An anti-apoptotic effect of AG in neuronal cells has recently been reported. However, whether there is a neuroprotective effect of unacylated ghrelin (UAG), the most abundant form of ghrelin in plasma, is still unknown. Therefore, we investigated whether UAG was neuroprotective against ischemic neuronal injury using primary cultured rat cortical neurons exposed to oxygen and glucose deprivation (OGD). Both AG and UAG inhibited OGD-induced apoptosis. Exposure of cells to the receptor-specific antagonist d-Lys-3-GHRH-6 abolished the protective effects of AG against OGD, whereas those of UAG were preserved, suggesting the involvement of a receptor that is distinct from GHS-R1a. Chemical inhibition of MAPK and phosphatidylinositol-3-kinase (PI3K) blocked the anti-apoptotic effects of AG and UAG. Ghrelin siRNA enhanced apoptosis either during OGD or even in normoxic conditions. The protective effects of AG and UAG were accompanied by an increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt, and glycogen synthase kinase-3β (GSK-3β). Furthermore, treatment of cells with AG or UAG resulted in nuclear translocation of β-catenin. In addition, both AG and UAG increased the Bcl-2/Bax ratio, prevented cytochrome c release, and inhibited caspase-3 activation. The data indicate that, independent of acylation, ghrelin can function as a neuroprotective agent that inhibits apoptotic pathways. These effects may be mediated via activation of the MAPK and PI3K/Akt pathways. Our data also suggest that PI3K/Akt-mediated inactivation of GSK-3β and stabilization of β-catenin contribute to the anti-apoptotic effects of ghrelin.

The neuronal damage caused by ischemic brain injury is associated with increased apoptosis. IGF-I exposure promotes neuronal defense and survival against ischemic insult by inhibiting apoptotic processes. We investigated the role of prostate apoptosis response-4 (Par-4), a proapoptotic gene the expression of which is increased after ischemic injury, in IGF-I-mediated inhibition of apoptosis using PC12 cells exposed to oxygen–glucose deprivation (OGD). The OGD insult resulted in significant increases in apoptotic cell death and Par-4 expression, which were prevented by the treatment of cells with an antisense oligonucleotide of Par-4. IGF-I treatment prior to OGD insult significantly reduced the number of apoptotic cells and the OGD-induced increase in Par-4 expression. OGD-induced nuclear translocation of Par-4 was also attenuated by IGF-I treatment. In addition, we demonstrated that the anti-apoptotic effect of IGF-I was blocked by chemical inhibition of a mitogen activated protien kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), or protein kinase A (PKA), but not by a protein kinase C inhibitor. Finally, pretreatment of cells with a MAPK or PI3K inhibitor attenuated IGF-I-induced inhibition of Par-4 expression, suggesting that the MAPK and PI3K pathways contribute to IGF-I-induced Par-4 suppression. In contrast, a PKA inhibitor failed to alter the inhibitory effect of IGF-I on Par-4. These findings indicate that in PC12 cells exposed to OGD insult, IGF-I protects cells from apoptosis, at least in part through the inhibition of Par-4 expression.

Ghrelin is an endogenous ligand for GH secretagogue receptor type 1a (GHSR1a), and is produced and released mainly from the stomach. It has been recently demonstrated that ghrelin can function as a neuroprotective factor by inhibiting apoptotic pathways. Kainic acid (KA), an excitatory amino acid l-glutamate analog, causes neuronal death in the hippocampus; previous studies suggest that activated microglia and astrocytes actively participate in the pathogenesis of KA-induced hippocampal neurodegeneration. However, it is unclear whether ghrelin has neuroprotective effect in KA-induced hippocampal neurodegeneration. I.p. injection of KA produced typical neuronal cell death in the CA1 and CA3 pyramidal layers of the hippocampus, and the systemic administration of ghrelin significantly attenuated KA-induced neuronal cell death in these regions through the activation of GHSR1a. Ghrelin prevents KA-induced activation of microglia and astrocytes, and the expression of proinflammatory mediators tumor necrosis factor α, interleukin-1β, and cyclooxygenase-2. The inhibitory effect of ghrelin on the activation of microglia and astrocytes appears to be associated with the inhibition of matrix metalloproteinase-3 expression in damaged hippocampal neurons. Our data suggest that ghrelin has a therapeutic potential for suppressing KA-induced pathogenesis in the brain.

Ghrelin, an endogenous ligand for the GH secretagogue receptor (GHS-R) receptor 1a (GHS-R1a), has been implicated in several physiologic processes involving the hippocampus. The aim of this study was to investigate the molecular mechanisms of ghrelin-stimulated neurogenesis using cultured adult rat hippocampal neural stem cells (NSCs). The expression of GHS-R1a was detected in hippocampal NSCs, as assessed by western blot analysis and immunocytochemistry. Ghrelin treatment increased the proliferation of cultured hippocampal NSCs assessed by BrdU incorporation. The exposure of cells to the receptor-specific antagonist d-Lys-3-GHRP-6 abolished the proliferative effect of ghrelin. By contrast, ghrelin showed no significant effect on cell differentiation. The expression of GHS-R1a was significantly increased by ghrelin treatment. The analysis of signaling pathways showed that ghrelin caused rapid activation of ERK1/2 and Akt, which were blocked by the GHS-R1a antagonist. In addition, ghrelin stimulated the phosphorylation of Akt downstream effectors, such as glycogen synthase kinase (GSK)-3β, mammalian target of rapamycin (mTOR), and p70^S6K. The activation of STAT3 was also caused by ghrelin treatment. Furthermore, pretreatment of cells with specific inhibitors of MEK/ERK1/2, phosphatidylinositol-3-kinase (PI3K)/Akt, mTOR, and Jak2/STAT3 attenuated ghrelin-induced cell proliferation. Taken together, our results support a role for ghrelin in adult hippocampal neurogenesis and suggest the involvement of the ERK1/2, PI3K/Akt, and STAT3 signaling pathways in the mediation of the actions of ghrelin on neurogenesis. Our data also suggest that PI3K/Akt-mediated inactivation of GSK-3β and activation of mTOR/p70^S6K contribute to the proliferative effect of ghrelin.