Ghrelin, an endogenous ligand for the GH secretagogue receptor was characterized recently from extracts of rat stomach. We describe the enteric distribution of ghrelin, ontogeny of stomach ghrelin gene expression, effects of dietary and endocrine manipulations, and vagotomy on stomach ghrelin mRNA and peptide levels and secretion in the rat. Ghrelin expression was examined by Northern blotting. Tissue and plasma ghrelin levels were measured by RIA. A gradient of ghrelin production occurs in the rat gastrointestinal tract with the highest ghrelin expression and peptide levels in the mucosal layer of the stomach-fundus and the lowest levels in the colon. Ghrelin was not detectable in the fetal stomach and increased progressively after birth especially during the second and third postnatal weeks. Plasma ghrelin levels also increased in parallel with stomach ghrelin levels postnatally. Exogenous GH treatment decreased stomach ghrelin expression significantly. A high-fat diet decreased plasma ghrelin levels, whereas a low-protein diet increased plasma ghrelin levels significantly. Intravenous administration of ghrelin stimulates gastrin and insulin secretion. Our findings indicate that ghrelin is an important stomach hormone sensitive to nutritional intake; ghrelin may link enteric nutrition with secretion of GH, insulin, and gastrin.

Apelin is a recently discovered peptide that is the endogenous ligand for the APJ receptor. The aim of this study was to characterize apelin expression (mRNA levels) in the rat gastrointestinal tract and pancreas, to localize distribution of apelin peptide-containing cells in the stomach by immunohistochemistry, and to characterize the ontogeny of gastric apelin expression and peptide and the influence of apelin on gastric cell proliferation in vitro. Additionally, the effect of apelin on cholecystokinin (CCK) secretion and the involvement of MAPK, protein kinase C, and changes in intracellular Ca(2+) in apelin-induced CCK secretion in vitro were examined. Northern analysis showed a maximal apelin expression in the stomach with a lower expression level in the intestine. Apelin expression was not detected in the pancreas. Immunohistochemistry revealed abundant apelin-positive cells in the glandular epithelium of the stomach. The ontogeny study showed a higher apelin expression in the fetal and postnatal rat stomachs when compared with the adult stomach. In contrast to apelin expression, apelin peptide was not detected in the rat stomach until 20 d of age and then increased progressively with age. Apelin was shown to stimulate gastric cell proliferation in vitro. Apelin also stimulated CCK secretion from a murine enteroendocrine cell line (STC-1); apelin-stimulated CCK secretion is mediated through MAPK but not by intracellular Ca(2+) signaling. Together, these data indicate that apelin is an important new stomach peptide with a potential physiological role in the gastrointestinal tract.

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Hematopoietic stem cell transplantation (HSCT) is used for treatment of lymphoma. In an attempt to design an efficacious and safe pre-HSCT conditioning regimen, we investigated the cytotoxicity of the combination of busulfan (B), melphalan (M) and gemcitabine (G) in lymphoma cell lines in the absence or presence of drugs that induce epigenetic changes. Cells were exposed to drugs individually or in combination and analyzed by the MTT proliferation assay, flow cytometry, and Western blotting. We used ~IC10 drug concentrations (57 μM B, 1 μM M and 0.02 μM G) which individually did not have major effects on cell proliferation. Their combination resulted in 50% inhibition of proliferation. Reduction to almost half concentration (20 μM B, 0.7 μM M and 0.01 μM G) did not have significant effects, but addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; 0.6 μM) to this combination resulted in a marked (~65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ATM-CHK2 pathway, phosphorylation of KAP1, epigenetic changes such as methylation and acetylation of histone 3, and activation of apoptosis. The relevance of epigenetic changes is further shown by the induction of DNA methyltransferases in tumor cells with low constitutive levels of DNMT3A and DNMT3B. The addition of 5-aza-2′-deoxycytidine (DAC) to [BMG+SAHA] further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by SAHA and DAC further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using [BMG+SAHA] as pre-HSCT conditioning for lymphoma.

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Dendritic spine development is crucial for the establishment of excitatory synaptic connectivity and functional neural circuits. Alterations in spine morphology and density have been associated with multiple neurological disorders. Autism candidate gene disconnected-interacting protein homolog 2 A (DIP2A) is known to be involved in acetylated coenzyme A (Ac-CoA) synthesis and is primarily expressed in the brain regions with abundant pyramidal neurons. However, the role of DIP2A in the brain remains largely unknown. In this study, we found that deletion of Dip2a in mice induced defects in spine morphogenesis along with thin postsynaptic density (PSD), and reduced synaptic transmission of pyramidal neurons. We further identified that DIP2A interacted with cortactin, an activity-dependent spine remodeling protein. The binding activity of DIP2A-PXXP motifs (P, proline; X, any residue) with the cortactin-Src homology 3 (SH3) domain was critical for maintaining the level of acetylated cortactin. Furthermore, Dip2a knockout (KO) mice exhibited autism-like behaviors, including excessive repetitive behaviors and defects in social novelty. Importantly, acetylation mimetic cortactin restored the impaired synaptic transmission and ameliorated repetitive behaviors in these mice. Altogether, our findings establish an initial link between DIP2A gene variations in autism spectrum disorder (ASD) and highlight the contribution of synaptic protein acetylation to synaptic processing.

Apelin is the endogenous ligand for the APJ receptor and both apelin and APJ are expressed in the gastrointestinal (GI) tract. The aim of this study was to define ontogeny of apelin and APJ in the developing rodent GI tract by measuring expression levels and characterizing abundance and cellular localization at an embryonic stage (E18.5 or E21), two postnatal stages (P4, P16) and in the adult. Apelin and APJ mRNA levels were measured by real time RT-PCR, apelin and APJ-containing cells were identified by immunohistochemical (IHC) staining. Gastric, duodenal and colonic apelin and APJ mRNA levels were highest at birth and declined postnatally. In the postnatal rat stomach, few apelin peptide-containing cells were identified, the density of gastric apelin-containing cells increased progressively after weaning and into adulthood. A robust APJ immunostaining was observed postnatally in the epithelium, intestinal goblet cells and in smooth muscle cells. In the adult rat, APJ immunostaining in the surface epithelium and goblet cells decreased markedly. During the early postnatal period, in an apelin-deficient mouse, APJ expression and immunostaining in the gut were reduced suggesting that apelin regulates APJ. Together, our data support a role for the apelin-APJ system in regulation of smooth muscle, epithelial and goblet cell function in the GI tract.

Apelin, a peptide widely expressed in the body, is the endogenous ligand for the APJ receptor. To investigate how the apelin gene is regulated transcriptionally, we cloned and characterized approximately 3000 and approximately 4000 bp 5'-upstream fragments of the rat and human apelin genes. Putative CAAT-like box, but not TATA-box sites were identified. The rat (-207/-1 bp) and human (-100/+74 bp) core promoter sequences contain putative binding sites for upstream stimulatory factor (USF)-1/-2. Mutagenesis and overexpression assays showed that USF up-regulates basal and inducible apelin transcription. EMSA and supershift experiments indicated binding of USF-1/-2 to the rat (-114/-109 bp) and human (-84/-79 bp) apelin promoters. ChIP experiments show that USF is recruited to the putative USF binding site in the human apelin promoter in cultured breast cells. In concert with increased breast apelin expression during pregnancy and lactation in rats, EMSAs demonstrate an elevated binding of pregnant and lactating rat breast nuclear proteins to a consensus USF oligonucleotide. In vivo ChIP assays verified increased USF binding to the apelin promoter in breast of lactating rats. Together, our findings show that USF exerts a stimulatory role in regulation of breast apelin expression during pregnancy and lactation.