The anti-inflammatory properties of COL-3 derives from its
ability to restrain glucose uptake and consequent glucose-dependent
NADPH production
Our data showed that the treatment with inflammogens LPS or αSa
substantially elevated glucose consumption by microglial cells, as it
seems to be a prerequisite to the shift into an activated profile
[43], estimated through the capture of [3H]-2-DG, a synthetic
analog of glucose labeled with tritium. This corroborates previous
results from our group showing LPS increases glucose uptake as a
substrate to NADPH-dependent ROS production, which leads to an
inflammatory state of microglial cells [32]. This is the first time
it is shown that the aggregated inflammatory form of αS can increase
glucose consumption in microglial cells, which points to a common
mechanism of these aggregated form of the protein with LPS to induce the
inflammatory state in microglial cells. Of interest, COL-3 strongly
inhibited [3H]-2-DG uptake in LPS- and
αSa-stimulated cells, indicating the non-antibiotic tetracycline
curtailed inflammatory-type responses by reducing glucose uptake. Again,
a higher concentration of the reference antibiotic tetracycline DOX
reproduced these findings; however, the microglial uptake of
[3H]-2-DG was unresponsive to DEX, indicating that
the mechanisms of action of the glucocorticoid and the tetracyclines
were most probably partly different.
It should be emphasized that the uptake of
[3H]-2-DG was also strongly reduced by inhibiting
NADPH oxidase activity with APO, which indicates that COL-3 prevented
the uptake of glucose through inhibition of ROS dependent-signaling
events [44]. Noticeably, COL-3 anti-inflammatory action was also
mirrored by unlabeled 2-DG used at concentrations that competitively
inhibit hexokinase, the glycolytic enzyme that converts glucose into
glucose-6-P [45], and saturating concentrations of GLUT-dependent
glucose uptake [46]. Our current data is one more evidence that the
control of glucose availability by anti-inflammatory drugs largely
contributes to the reduction of microglial cell activation [32,47].
NADPH oxidase (NOX2), a multimeric complex, utilizes NADPH as substrate
and reduces molecular oxygen to produce superoxide, that is then further
dismutated into stable and diffusible hydrogen peroxide pro-oxidant
[48]. The first metabolite product of glucose, glucose-6-phosphate,
can be translocated into the pentose phosphate shunt [49], which
serves to produce nucleotide precursors and helps regenerate the
reducing agent NADPH, which is also a requisite substrate for NOX2
[45,49]. Consistent with previous findings from our group and
others, the pentose-phosphate shunt pathway is essential for ROS
response in immune cells like macrophages and microglial cells
[32,48]. Therefore, we tested the possibility that the increase in
glucose uptake observed in primary microglial cells may serve to
stimulate an increase in NADPH levels and that COL-3 may possibly
resolve inflammatory processes by preventing this effect. Here we
observed a robust elevation of NADPH synthesis in LPS- and
αSa-challenged microglial cells.
In a recent study from our group, the phytocannabinoid anti-inflammatory
cannabidiol prevented the rise in NADPH synthesis elicited by LPS
treatment, indicating that the anti-inflammatory action of cannabidiol
may be also dependent of this suppressive effect [32]. Our current
results sustain our previous data, as the tetracyclines COL-3 and DOX
reduced NADPH levels produced after LPS and αSa stimulation in
microglial cells. Like cannabidiol, COL-3 operated by interfering with
an alternative glucose-dependent mechanism. Therefore, it is possible to
imply that the inhibitory effects that COL-3 exerts on glucose
consumption and NADPH synthesis may strengthen the intrinsic antioxidant
potential of this compound in a self-reinforcing process.
Reinforcing our hypothesis, we found that compounds capable of
restricting LPS and αSa-induced TNF-α release and Iba-1 expression were
also effective in preventing not only [3H]-2-DG
uptake, but also the consequent production of intracellular NADPH
derived from the preferential activation of the pentose-phosphate shunt
pathway. In this way, the NOX2 inhibitor APO and the saturating
concentration of unlabeled 2DG prevented the production of NADPH,
confirming the resulting inflammatory process caused by these
inflammogens results from a signaling event that requires an increase in
glucose uptake and consequent NADPH production, and the
anti-inflammatory effect of COL-3 comes from blocking the signaling
cascade of the pentose-phosphate shunt pathway.
Our results also show that DEX, despite reducing inflammatory factors,
was ineffective in avoiding LPS- and αSa-dependent
[3H]-2AG uptake increase. Although there are
reports showing DEX inhibits glucose uptake in contracting myotubes
[50], glucocorticoids classically suppress the transcription of
several genes that encode pro-inflammatory cytokines and chemokines,
therefore suggesting it may not have a direct action over glucose uptake
and consumption and oxidative stress response.