innate immune cells, hence furthering a pro-inflammatory plaque
microenvironment [5, 6].
Macrophages are dynamic innate immune cells that have the
tremendous ability to store, mobilize, and efflux cholesterol. There
are numerous pathways that contribute to the process of exporting
esterified cholesterol (stored as cholesteryl esters; CE); however,
the ATP-binding cassette transporters, ABCA1 and ABCG1, are
the main transport proteins that facilitate the efflux of free choles-
terol to lipid-poor apolipoprotein A-1 (apoA-I) and high-density
lipoprotein (HDL), respectively [7–9]. Macrophage cholesterol
efflux, in the context of atherosclerosis, represents the first step in
reverse cholesterol transport (RCT), the physiological process
where cholesterol is removed from peripheral tissue for eventual
recycling or elimination. This process is anti-atherogenic and has
garnered intense therapeutic interest [10–13]. This chapter focuses
on methods that are critical for monitoring macrophage cholesterol
synthesis, uptake, and efflux and that have shed light on the impor-
tance of AMP-activated protein kinase (AMPK) in the past.
Playing a fundamental role in regulating energy metabolism,
AMPK lies at the nexus of macrophage lipid homeostasis and
inflammation. In macrophages (and most other myeloid-derived
cells) AMPK is expressed exclusively as a α 1 β 1 γ1 heterotrimer
[14]. Although the immunometabolic aspects of AMPK have
been recently reviewed [15–17], some of the first evidence linking
AMPK to a protective role in atherosclerosis used a macrophage cell
line with the indirect and non-specific activator 5-aminoimidazole-
4-carboxamide ribonucleotide (AICAR) [18]. The first in vivo evi-
dence linking macrophage AMPK to a protection against athero-
genesis used a myeloid AMPKα1 knockout crossed to the
atherosusceptible LDL receptor knockout mouse, which demon-
strated that myeloid AMPK regulates macrophage migration, adhe-
sion, and ultimately content within atherosclerotic plaque,
functions above and beyond some of the classically described roles
[19]. However, the protective role for myeloid AMPK in athero-
sclerosis was recently challenged when myeloid AMPKα1 knock-
out mice were crossed to the apolipoprotein E (apoE)-deficient
atherogenic background. In this model, AMPKα1 was shown to
promote monocyte to macrophage differentiation, and the deletion
of AMPK was linked to a defect in autophagy that diminished the
number of plaque macrophages [20]. Although there are technical
variations between the various in vivo publications, the role for
macrophage AMPK in atherosclerosis remains unclear [19–22].
Atherosclerosis represents a complex chronic disease whose
initiation and progression requires and affects numerous different
cell types, and AMPK has been the focus of many non-macrophage
investigations [15]. However, we recently reported that AMPK can
regulate numerous aspects of macrophage cholesterol homeostasis
in primary mouse macrophage, and moreover, activation of AMPK478 Nicholas D. LeBlond and Morgan D. Fullerton