We previously established a rat model of diabetic cardiomyopathy (DCM) and found that the expression of lncRNA H19 was significantly downregulated. The present study was designed to investigate the pathogenic role of H19 in the development of DCM. Overexpression of H19 in diabetic rats attenuated oxidative stress, inflammation and apoptosis, and consequently improved left ventricular function. High glucose was associated with reduced H19 expression and increased cardiomyocyte apoptosis. To explore the molecular mechanisms involved, we performed in vitro experiments using cultured neonatal rat cardiomyocytes. Our results showed that miR-675 expression was decreased in cardiomyocytes transfected with H19 siRNA. The 3′UTR of VDAC1 was cloned downstream of a luciferase reporter construct and cotransfected into HEK293 cells with miR-675 mimic. The results of luciferase assay indicated that VDAC1 might be a direct target of miR-675. The expression of VDAC1 was upregulated in cardiomyocytes transfected with miR-675 antagomir, which consequently promotes cellular apoptosis. Moreover, enforced expression of H19 was found to reduce VDAC1 expression and inhibit apoptosis in cardiomyocytes exposed to high glucose. In conclusion, our study demonstrates that H19/miR-675 axis is involved in the regulation of high glucose-induced apoptosis by targeting VDAC1, which may provide a novel therapeutic strategy for the treatment of DCM.

Human chromosome 1p35-p36 has long been suspected to harbor a tumor suppressor gene in pancreatic cancer and other tumors. We found that expression of rap1GAP, a gene located in this chromosomal region, is significantly downregulated in pancreatic cancer. Only a small percentage of preneoplastic pancreatic intraductal neoplasia lesions lost rap1GAP expression, whereas loss of rap1GAP expression occurred in 60% of invasive pancreatic cancers, suggesting that rap1GAP contributes to pancreatic cancer progression. In vitro and in vivo studies showed that loss of rap1GAP promotes pancreatic cancer growth, survival, and invasion, and may function through modulation of integrin activity. Furthermore, we showed a high frequency of loss of heterozygosity of rap1GAP in pancreatic cancer. Collectively, our data identify rap1GAP as a putative tumor suppressor gene in pancreatic cancer. (Cancer Res 2006; 66(2): 898-906)

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2′- O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2′- O-Me-cAMP-AM was associated with depolarization and an increase of [Ca2+]i that reflected both Ca2+ influx and intracellular Ca2+ mobilization in islet β-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2′- O-Me-cAMP-AM (10 μM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2′- O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca2+-dependent exocytosis.

Epac1 and Epac2 are guanine nucleotide exchange factors activated by adenosine-3Ј,5Ј-cyclic monophosphate (cAMP), and which are known to be expressed in numerous mammalian cell types (1, 2). An accumulating body of evidence indicates that the existence of Epac may explain novel protein kinase A (PKA) 2 independent actions of cAMP that underlie cellular responsiveness to hormones, neurotransmitters, and pharmacological agents of therapeutic importance (3). Selective activation of Epac may be achieved through the use of 8-(4-chlorophenylthio)-2Ј-O-methyladenosine-3Ј,5Ј-cyclic monophosphate, also known as 8-pCPT-2Ј-O-Me-cAMP (4, 5). This cAMP analog, which incorporates a 2Ј-O-methyl group on the ribose ring of the nucleotide, as well as a 4-chlorophenylthio group on position 8 of the adenine moiety, acts as a "superactivator" of Epac while having a greatly diminished ability to activate PKA (4). Thus, 8-pCPT-2Ј-O-Me-cAMP is an Epac-selective cAMP analog (ESCA) (6).8-pCPT-2Ј-O-Me-cAMP can cross the plasma membrane and is able to alter diverse cellular functions that include Rap1 GTPase activity, PKB, and ERK1/2 protein kinase activity, phospholipase C⑀ activity, Ca 2ϩ signaling, ion channel activity, exocytosis, cell adhesion, and gene expression (7-9). Although no selective antagonist of Epac activation exists, these effects of 8-pCPT-2Ј-O-Me-cAMP are believed to be Epac-mediated because they are observed under conditions in which PKA activity is blocked, whereas they are reduced or eliminated when Epac gene expression is down-regulated. Furthermore, such actions of 8-pCPT-2Ј-O-Me-cAMP are measurable in cells that do not express the cyclic nucleotide-regulated ion channels that constitute an alternative target of cAMP action.Interestingly, published findings exist in which cAMP-elevating agents were found to exert actions not attributable to * This work was supported, in whole or in part, by National Institutes of Health Grants DK045817 and DK069575 (to G. G. H.). This work was also supported by a American Diabetes Association Research Award (to C. A. L. -89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; Luc, luciferase; PDE, cyclic nucleotide phosphodiesterase; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; SES, standard extracellular saline; RIP1, rat insulin 1 gene promoter; Epac, exchange protein directly activated by cAMP.

Forebrain neurogenesis persists throughout life in the rodent subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Several strategies have been employed to eliminate adult neurogenesis and thereby determine whether depleting adult-born neurons disrupts specific brain functions, but some approaches do not specifically target neural progenitors. We have developed a transgenic mouse line to reversibly ablate adult neural stem cells and suppress neurogenesis. The nestin-tk mouse expresses herpes simplex virus thymidine kinase (tk) under the control of the nestin 2nd intronic enhancer, which drives expression in neural progenitors. Administration of ganciclovir (GCV) kills actively dividing cells expressing this transgene. We found that peripheral GCV administration suppressed SVZ-olfactory bulb and DG neurogenesis within two weeks but caused systemic toxicity. Intracerebroventricular GCV infusion for 28 days nearly completely depleted proliferating cells and immature neurons in both the SVZ and DG without systemic toxicity. Reversibility of the effects after prolonged GCV infusion was slow and partial. Neurogenesis did not recover 2 weeks after cessation of GCV administration, but showed limited recovery 6 weeks after GCV that differed between the SVZ and DG. Suppression of neurogenesis did not inhibit antidepressant responsiveness of mice in the tail suspension test. These findings indicate that SVZ and DG neural stem cells differ in their capacity for repopulation, and that adult-born neurons are not required for antidepressant responses in a common behavioral test of antidepressant efficacy. The nestin-tk mouse should be useful for studying how reversible depletion of adult neurogenesis influences neurophysiology, other behaviors, and neural progenitor dynamics.