技术资料

Incubation of skeletal muscle with 5-aminoimidazole-4carboxamide ribonucleoside (AICAR),a compound that activates 5'-AMP-activated protein kinase (AMPK),has been demonstrated to stimulate glucose transport and GLUT4 translocation to the plasma membrane. In this study,we characterized the AMPK cascade in 3T3-L1 adipocytes and the response of glucose transport to incubation with AICAR. Both isoforms of the catalytic alpha-subunit of AMPK are expressed in 3T3-L1 adipocytes,in which AICAR stimulated AMPK activity in a time- and dose-dependent fashion. AICAR stimulated 2-deoxy-D-glucose transport twofold and reduced insulin-stimulated uptake to 62% of the control transport rate dose-dependently,closely correlating with the activation of AMPK. AICAR also inhibited insulin-stimulated GLUT4 translocation,assessed using the plasma membrane lawn assay. The effects of AICAR on insulin-stimulated glucose transport are not mediated by either adenosine receptors or nitric oxide synthase and are mediated downstream of phosphatidylinositol 3'-kinase stimulation. We propose that in contrast to skeletal muscle,in which AMPK stimulation promotes glucose transport to provide ATP as a fuel,AMPK stimulation inhibits insulin-stimulated glucose transport in adipocytes,inhibiting triacylglycerol synthesis,to conserve ATP under conditions of cellular stress. Investigation of the mode of action of AICAR and AMPK may,therefore,give insight into the mechanism of insulin action. View Publication

The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways,the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidazole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK,i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose,heat shock),AICAR does not perturb the cellular contents of ATP,ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation,causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase),and almost total cessation of two of the known target pathways,i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase,previously demonstrated in cell-free assays,also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade. View Publication

Molecular mechanisms of how energy metabolism affects embryonic stem cell (ESC) pluripotency remain unclear. AMP-activated protein kinase (AMPK),a key regulator for controlling energy metabolism,is activated in response to ATP-exhausting stress. We investigated whether cellular energy homeostasis is associated with maintenance of self-renewal and pluripotency in mouse ESCs (mESCs) by using 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) as an activator of AMPK. We demonstrate that AICAR treatment activates the p53/p21 pathway and markedly inhibits proliferation of R1 mESCs by inducing G(1) /S-phase cell cycle arrest,without influencing apoptosis. Treatment with AICAR also significantly reduces pluripotent stem cell markers,Nanog and stage-specific embryonic antigen-1,in the presence of leukemia inhibitory factor,without affecting expression of Oct4. H9 human ESCs also responded to AICAR with induction of p53 activation and repression of Nanog expression. AICAR reduced Nanog mRNA levels in mESCs transiently,an effect not due to expression of miR-134 which can suppress Nanog expression. AICAR induced Nanog degradation,an effect inhibited by MG132,a proteasome inhibitor. Although AICAR reduced embryoid body formation from mESCs,it increased expression levels of erythroid cell lineage markers (Ter119,GATA1,Klf1,Hbb-b,and Hbb-bh1). Although erythroid differentiation was enhanced by AICAR,endothelial lineage populations were remarkably reduced in AICAR-treated cells. Our results suggest that energy metabolism regulated by AMPK activity may control the balance of self-renewal and differentiation of ESCs. View Publication

5-Azacytidine was first synthesized almost 40 years ago. It was demonstrated to have a wide range of anti-metabolic activities when tested against cultured cancer cells and to be an effective chemotherapeutic agent for acute myelogenous leukemia. However,because of 5-azacytidine's general toxicity,other nucleoside analogs were favored as therapeutics. The finding that 5-azacytidine was incorporated into DNA and that,when present in DNA,it inhibited DNA methylation,led to widespread use of 5-azacytidine and 5-aza-2'-deoxycytidine (Decitabine) to demonstrate the correlation between loss of methylation in specific gene regions and activation of the associated genes. There is now a revived interest in the use of Decitabine as a therapeutic agent for cancers in which epigenetic silencing of critical regulatory genes has occurred. Here,the current status of our understanding of the mechanism(s) by which 5-azacytosine residues in DNA inhibit DNA methylation is reviewed with an emphasis on the interactions of these residues with bacterial and mammalian DNA (cytosine-C5) methyltransferases. The implications of these mechanistic studies for development of less toxic inhibitors of DNA methylation are discussed. View Publication