title: Overexpression of AMP-metabolizing enzymes controls adenine nucleotide levels and AMPK activation in HEK293T cells.
abstract: We investigated whether overexpression of AMP-metabolizing enzymes in intact cells would modulate oligomycin-induced AMPK activation. Human embryonic kidney (HEK) 293T cells were transiently transfected with increasing amounts of plasmid vectors to obtain a graded increase in overexpression of AMP-deaminase (AMPD) 1, AMPD2, and soluble 5'-nucleotidase IA (cN-IA) for measurements of AMPK activation and total intracellular adenine nucleotide levels induced by oligomycin treatment. Overexpression of AMPD1 and AMPD2 slightly decreased AMP levels and oligomycin-induced AMPK activation. Increased overexpression of cN-IA led to reductions in the oligomycin-induced increases in AMP and ADP concentrations by ∼70 and 50%, respectively, concomitant with a 50% decrease in AMPK activation. The results support the view that a rise in ADP as well as AMP is important for activation of AMPK, which can thus be regulated by the adenylate energy charge. The control coefficient of cN-IA on AMP was 0.3-0.7, whereas the values for AMPD1 and AMPD2 were <0.1, suggesting that in this model cN-IA exerts a large proportion of control over intracellular AMP. Therefore, small molecule inhibition of cN-IA could be a strategy for AMPK activation.
authors.affiliation: Université Catholique de Louvain and de Duve Institute, Brussels, Belgium.
authors.forename: Catheline
authors.initials: C
authors.lastname: Plaideau
authors.forename: Jianming
authors.initials: J
authors.lastname: Liu
authors.forename: Judith
authors.initials: J
authors.lastname: Hartleib-Geschwindner
authors.forename: Laurent
authors.initials: L
authors.lastname: Bastin-Coyette
authors.forename: Françoise
authors.initials: F
authors.lastname: Bontemps
authors.forename: Jan
authors.initials: J
authors.lastname: Oscarsson
authors.forename: Louis
authors.initials: L
authors.lastname: Hue
authors.forename: Mark H
authors.initials: MH
authors.lastname: Rider
mesh_terms: D015720:5'-Nucleotidase; D000659:AMP Deaminase / Q000378:metabolism; D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D009840:Oligomycins / Q000494:pharmacology
chemical_list: D000227:Adenine Nucleotides; D009840:Oligomycins; D000249:Adenosine Monophosphate; D055372:AMP-Activated Protein Kinases; D015720:5'-Nucleotidase; D000659:AMP Deaminase; C514117:AMPD1 protein, human; C475566:AMPD2 protein, human