LDL-cholesterol, leading to the formation of “foam cells” and
development of atherosclerotic plaque [20]. The degree of mono-
cyte adhesion in response to test substances can be evaluated in
culture by exposing endothelial cells to pro-inflammatory cytokines
in the presence or absence of AMPK activators, which are then
washed away prior to incubation with a pro-monocytic cell line.
Monocytes which are not tightly attached to the endothelial cells
are then washed away, before the adherent monocytes are fixed and
counted (Fig.3). These techniques provide multiple methods with
which to investigate the role of AMPK in inflammatory pathways in
cultured or isolated cells.2 Materials
AMPK activation can be achieved pharmacologically using direct
activators such as A769662 and Compound 991 or the
AMP-mimetic AICAR [21]. A769662 and Compound 991 are
poorly soluble in aqueous solution, yet are soluble in DMSO,Fig. 2NFκB nuclear localization in adipocytes overexpressing constitutively active AMPKα1. 3T3-L1
adipocytes which overexpress the coxsackievirus and adenovirus receptor (3T3-L1Δ1CAR) [12, 23] were
infected with 100 pfu/cell adenoviruses expressing GFP (Ad-GFP) or constitutively active AMPK-α1 (Ad-AMPK-
CA) (which expresses GFP from a separate promotor) [12, 24] for 48 h prior to stimulation with IL-1β(10 ng/
ml) for 15 min. NFκB nuclear localization was assessed by confocal microscopy
Roles in Inflammation 309