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Page 154 Harry et al. Neuroimmunol Neuroinflammation 2020;7:150-65 I http://dx.doi.org/10.20517/2347-8659.2020.07

the Krebs cycle and OXPHOS remain intact. In addition, the cells are able to utilize fatty acid oxidation
and oxidative respiration for energy production [98,123-125] and arginine metabolism is shifted to ornithine
and polyamines [126] . Work by Ferger et al. [118] suggested that the stability of the electron transport chain
in mitochondria plays a more substantial and critical role for the microglia response to IL-4 as compared
to the response to LPS. In microglia, exposure to LPS induced a rapid and transitory decrease in the
mitochondrial uncoupling protein-2 (UCP-2) levels accompanied by increased mitochondrial reactive
oxygen species (mtROS) production. In UCP-2-silenced microglia, the response to LPS was exacerbated
and a response to IL-4 was eliminated [127] . An earlier study examining the translation of responses in
macrophages to microglia reported reduced glucose consumption and lactate production in BV-2 cells
exposed to IL-4 [109] . It was suggested that this phenotype was associated with phagocytosis of debris and
[69]
the reduced need for anabolic reactions. Similar findings were reported by Orihuela et al. with exposure
of BV-2 cells or primary murine microglia to IL-4/IL-13 in that the cells remained within an oxidative
metabolic state with OCR and ECAR levels similar to non-stimulated cells. There were also elevations in
[69]
mRNA levels for Ym1, Il4, Cd163, and Arg1, but no induction of Tnfa or Il1 . The lack of a demonstrated
metabolic shift with IL-4/IL-13 stimulation is in contrast to observations in peripheral macrophages of
stimulated glucose uptake in addition to fatty acid metabolism and shift in mitochondrial biogenesis [125] .

MITOCHONDRIA AND FREE RADICAL PRODUCTION
A key feature of classically activated macrophages is their ability to produce reactive oxygen species (ROS)
to facilitate killing of phagocytized bacteria [128] . Stimulation of macrophages with LPS and IFN-g increases
inducible nitric oxide synthase (iNOS), generating nitric oxide (NO), a reactive nitrogen species that
can inhibit mitochondrial respiration by nitrosylating iron-sulfur proteins in electron transport chain
complexes and cytochrome c oxidase [129,130] . It is considered that iNOS and NO-mediated inhibition of
mitochondrial metabolism in macrophages is essential for the metabolic switch activated by LPS. This is
not as well established in microglia, especially given that, while nitric oxide production is often linked with
pro-inflammatory cytokines, such cytokines can be stimulated by sterile activators in the absence of NO. In
microglia, it has been proposed that activation of the rapamycin (mTOR) pathway may actively contribute
to this process [131] as well as pro-inflammatory cytokine production and phagocytic activity [132-134] . The
resulting elevated thiamin pyrophosphate activity increased production of purines and pyrimidines, which
yield nicotinamide adenine dinucleotide phosphate (NADPH) for the NADPH oxidase enzyme and ROS
production [135] implicated in the transition of microglia to a pro-inflammatory phenotype [136-139] . It has
been proposed that glycolytic ATP production may utilize the electron transport chain to compensate
for this shift towards ROS production [128] . It is known that superoxide produced by NADPH oxidase
is predominantly extracellular. In vivo, extracellular superoxide dismutase 3 (SOD3) forms membrane
permeable H O . Studies have suggested that H O , rather than SOD, serves as the primary ROS involved
2
2
2
2
in mediating microglial activation and proliferation in response to pro-inflammatory stimuli [136,140,141] . H O
2
2
has also been implicated in the increase in CD11b expression both in vitro and in vivo [142] , as well as in
persistent neuroinflammation related to impaired NF-kB p50 function [143] . Superoxide anion is the primary
ROS produced by mitochondria and mitochondria-derived H O and, in addition to NADPH oxidase, may
2
2
contribute to a pro-inflammatory phenotype of microglia such as that observed with the mitochondrial
toxin, rotenone [144] . With a response sufficient to result in ROS production, the associated intracellular
damage is limited by increased generation of NADPH required for maintenance of reduced glutathione and
nitric oxide production [145,146] .
GLUCOSE
In addition to the critical role that glucose plays in energy metabolism, it serves as an exclusive substrate
for the hexose monophosphate shunt, which produces NADPH that is required by glutathione reductase to
convert oxidized glutathione (GSSG) back to reduced glutathione (GSH). It also serves to quench ROS and