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.

Interplay between adenylate metabolizing enzymes and AMP‐activated protein kinase

“…In contrast, the involvement of AMPD2 and AMPD3, expressed mainly in the liver and erythrocytes, respectively, has been largely overlooked. In HEK293T cells, overexpression of both AMPD1 and AMPD2 slightly decreased AMP levels and oligomycin‐induced AMPK activation . Recently, AMPD2 has been implicated in mediating the toxic effects of high fructose intake .…”

The Role of Adenosine Signaling in Headache: A Review

“…Mitochondrial dysfunction and adenosine signaling could affect pain via activation of AMPK, a protein kinase heavily associated with pain plasticity [93]. Extracellular adenosine, increases in the AMP:ATP ratio during hypoxia, and inhibition of 5′NT all enhance AMPK activity [94,95,96,97]. It is possible that mitochondrial dysfunction within trigeminal pain processing brain regions exacerbates adenosinergic mechanisms and AMPK activity to produce headache.…”

Role of AMP-activated protein kinase in metabolic depression in animals

“…This rise in free AMP upon flight would be more than sufficient to activate AMPK (Pan and Hardie 2002); however, one should bear in mind that AMP concentrations required for activation increase in the presence of free ATP (not complexed with Mg 2+ ) due to competitive binding of the nucleotides to the sites on the AMPKγ regulatory subunits (Gowans et al 2013). Ideally, calculations of free AMP concentrations should be made after measurements of total nucleotides in tissues, as reported in hypoxic goldfish and turtle (Jibb and Richards 2008;Galli et al 2013), and quantification of total nucleotide concentrations by HPLC (Plaideau et al 2012; would be more reliable than enzymatic determinations. Fig.…”

“…It is possible that AMPK might be activated on arousal from hibernation, when fatty acid oxidation is needed for thermogenesis. We showed previously that AMPK activation can be modulated by changes in the activity of AMP-deaminase (AMPD), the AMP-metabolizing enzyme that converts AMP to IMP (Plaideau et al 2012;, and kinetic data suggested that AMPD would be inhibited in muscle of the white-tailed prairie dog during hibernation (English and Storey 2000). A decrease in AMPD2 activity seen in liver could contribute to AMPK activation during hibernation (Lanaspa et al 2015).…”